Method for freeze-drying cells in a hydrogel comprising nanofibrillar cellulose and freeze-dried cells in an aerogel comprising nanofibrillar cellulose

ABSTRACT

The present disclosure provides a method for freeze-drying cells in a hydrogel comprising nanofibrillar cellulose, the method comprising providing a hydrogel comprising nanofibrillar cellulose, providing cells, combining the cells and the hydrogel comprising nanofibrillar cellulose to form a cell system, and freeze drying the cell system to obtain dried cells in a hydrogel comprising nanofibrillar cellulose. The present disclosure also provides a freeze-dried hydrogel comprising nanofibrillar cellulose and cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional application of U.S. application Ser. No.16/468,390, filed Jun. 11, 2019, which is a National Stage applicationof PCT/EP2017/071441, filed Aug. 25, 2017, which claims benefit ofEuropean Application No. 16397537.8 filed on Dec. 15, 2016, each ofwhich is incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present disclosure relates to methods for freeze-drying cells, andfreeze-dried cells obtained with said method. More particularly thepresent disclosure relates to methods for freeze-drying cells in ahydrogel comprising nanofibrillar cellulose and to freeze-dried cells inan aerogel comprising nanofibrillar cellulose

BACKGROUND

With current knowledge, freeze-drying of complex or large mammaliancells causes irreversible damage to the cell and the freeze-dried cellstend to lose their three-dimensional shape when dried. Therefore thereis need to find a method which may be used for freeze-drying such cells,and which could protect the cells during freezing.

The obtained dried product should be dry, active, shelf stable, cleanand sterile, ethically acceptable, pharmaceutically elegant, readilysoluble and simple to reconstitute, and the process should beeconomically practicable.

SUMMARY

Nanofibrillar cellulose refers to isolated cellulose fibrils or fibrilbundles derived from cellulose raw material. Nanofibrillar cellulose isbased on a natural polymer that is abundant in nature. Nanofibrillarcellulose has a capability of forming viscous hydrogel in water.

Nanofibrillar cellulose production techniques are based on grinding ofaqueous dispersion of pulp fibers. The concentration of nanofibrillarcellulose in dispersions is typically very low, usually around 0.3-5%.After the grinding or homogenization process, the obtained nanofibrillarcellulose material is a dilute viscoelastic hydrogel.

Because of its nanoscale structure nanofibrillar cellulose has uniqueproperties which enable functionalities which cannot be provided byconventional cellulose. It was found out that cells, especiallyeukaryotic cells, may be grown in nanofibrillar cellulose matrix inthree-dimensional culture, wherein the cells may grow as spheroids. Suchcell cultures are efficient, as the cell in the 3D culture cancommunicate easily with each other and form tissue-like colonies. Thisis not possible by using conventional 2D cultures.

However, because of the nanoscale structure nanofibrillar cellulose isalso a challenging material. For example dewatering or handling ofnanofibrillar cellulose may be difficult. Further, after dewatering itis generally difficult to rehydrate or regel the dried material toobtain material having equal properties to the original nanofibrillarcellulose before the dewatering or drying. Especially challengingdewatering process is freeze-drying.

In the present application it is disclosed how to use the nanofibrillarcellulose hydrogel as a lyoprotective matrix for freeze-drying cells,such as mammalian cells. It has been a problem how nanofibrillarcellulose (NFC) hydrogel as such can be freeze-dried and then rehydratedwith no loss in rheological or diffusive properties. For example, thedrying of NFC promotes irreversible hydrogen bonding between neighboringNFC nanofibers, known as hornification. However, when cells werefreeze-dried in nanofibrillar cellulose hydrogel, the hydrogel itselfwas found to provide lyoprotective properties. When 2D cultures, evenwith trehalose, were lyophilized, the cells did not survive. It was alsofound out that specific auxiliary agents may to be used to furtherprotect the hydrogels and the cells during freezing and lyophilization.

One embodiment provides a method for freeze-drying cells in a hydrogelcomprising nanofibrillar cellulose, the method comprising

-   -   providing a hydrogel comprising nanofibrillar cellulose,    -   providing cells,    -   combining the cells and the hydrogel comprising nanofibrillar        cellulose to form a cell system, and    -   freeze drying the cell system to obtain dried cells in an        aerogel comprising nanofibrillar cellulose.

One embodiment provides a method for freeze-drying extracellularvesicles in a hydrogel comprising nanofibrillar cellulose, the methodcomprising

-   -   providing a hydrogel comprising nanofibrillar cellulose,    -   providing extracellular vesicles,    -   combining the vesicles and the hydrogel comprising nanofibrillar        cellulose to form a vesicle system, and    -   freeze drying the vesicle system to obtain dried extracellular        vesicles in an aerogel comprising nanofibrillar cellulose.

The methods may comprise providing one or more cryoprotective and/orlyoprotective agent(s) and adding the one or more cryoprotective and/orlyoprotective agent(s) to the cell system or to the vesicle system.

One embodiment provides a freeze-dried aerogel comprising nanofibrillarcellulose, cells and optionally one or more cryoprotective and/orlyoprotective agent(s), wherein the moisture content of the freeze-driedaerogel is 10% or less, preferably in the range of 1-10% (w/w), such as2-8% (w/w).

One embodiment provides a freeze-dried aerogel comprising nanofibrillarcellulose and extracellular vesicles, wherein the moisture content ofthe freeze-dried aerogel is 10% or less, preferably in the range of1-10% (w/w), such as 2-8% (w/w).

The main embodiments are characterized in the independent claims.Various embodiments are disclosed in the dependent claims. Theembodiments recited in claims and in the embodiments are mutually freelycombinable unless otherwise explicitly stated.

It was surprisingly found out that nanofibrillar cellulose hydrogelcould act as a lyoprotective matrix in a freeze-drying process. In theprocess ingredients may be included in the hydrogel. The ingredients mayinclude cells, such as eukaryotic or prokaryotic cells, especiallymammalian cells, but also other agents, such as cryo- and/orlyoprotective agents, pharmaceutical or therapeutical agents, nutrients,other chemical agents, active agents or other large or small molecules.However, it was specifically found out that the nanofibrillar cellulosehydrogel as such provided efficient lyoprotective properties for cells.This is advantageous because especially eukaryotic cells, moreparticularly mammalian cells, are challenging to freeze-dry. One reasonfor this could be the mitochondria or vesicles present in the cells,which are easily damaged during the process.

It was possible to obtain a dried product, which could be rehydrated orredispersed into a form which restores the original properties of thehydrogel comprising nanofibrillar cellulose, i.e. the dried product canbe regelled. Such properties include for example cell properties, gelproperties and controlled release of active compounds or pharmaceuticalingredients.

Nanofibrillar cellulose was discovered to act as a lyoprotective matrixthat can withstand freezing and drying without mechanical rupture.Nanofibrillar cellulose may also act as a cryoprotective matrix. It isinert, clean of contaminants, holds enough liquid to achieve highporosity and can be shaped as well-defined geometrical units. Further,the nanofibrillar cellulose is able to release absorbed active productswhen flooded with rehydration liquid. To release cell, usually alsoenzyme addition is required to degrade the gel.

It was discovered that the cells retained their original morphology andno ruptures or collapsing was detected. Further, the rehydrated cellsattached to the surface of the culture flask, which indicates theviability of the cells. The lyoprotective effects of the nanofibrillarcellulose hydrogel were demonstrated in tests.

Further, it was discovered that extracellular vesicles, such asmicrovesicles, could survive the freeze-drying of the cells when themethod described herein was used. As microvesicles have a role inintercellular messaging, the preservation of microvesicles during thefreeze-drying enhances the ability of the cell culture to start growingefficiently as a 3D colony immediately after rehydration. It is possibleto freeze-dry also separate microvesicles with the method.

The nanofibrillar cellulose hydrogel provides a hydrophilic matrix,which is non-toxic, biocompatible and also biodegradable. For examplethe matrix can be degraded enzymatically, for example by addingcellulase. On the other hand the hydrogel is stable at physiologicalconditions. Cells, especially challenging eukaryotic cells, such asmammalian cells, can be cultured in nanofibrillar cellulose hydrogel inthree-dimensional cultures, freeze-dried, and rehydrated to restore the3D culture.

The feature that the hydrogel can be enzymatically digested, isadvantageous especially is the case of cells. When a cell system isdried, its biological clock stops; similarly, when frozen. Therefore, acompletely new generation of “just-add-water” cell products may beprovided, where the supportive dry NFC matrix could be removed aftertransportation. This may in return accelerate the cell research, as theresearch typically requires a lot of currently irreplaceable handwork.Also, transporting and storing dried ready-to-use cell systems would bemore affordable, as complex cooling systems would not be required. Forexample 3D cultivated cell spheroid products could be made directlyaccessible to researchers without the requirement to seed and grow themfirst. As 3D spheroids better mimic real tumors, particularly forexample if hypoxia is considered, their usage will likely increase inthe near future. Later, clinical applications for the freeze-dried cellproducts could be considered in a more serious manner.

Further, the presence of the selected cryo- and/or lyoprotectants had noeffect to the release profile of the agents from the gels. By drying thehydrogel it is possible to obtain a very long shelf life for medical orscientific products. Especially gels containing cells or active agents,which are unstable at moist conditions, such as proteins, DNA/RNA, andother agents sensitive to hydrolysis, can be successfully freeze-driedinto forms containing little or practically no water and thereforehaving a prolonged stability and shelf life. Such freeze-dried productsmay be stored even at room temperature and may be regelled prior to useby adding liquid, such as water, diluted saline or diluted cell culturemedium.

Certain advantageous properties of the hydrogel comprising nanofibrillarcellulose include flexibility, elasticity and remouldability. As thehydrogel contains a lot of water, it may also show good permeability.The hydrogels of the embodiments also provide high water retentioncapacity and molecule diffusion property speed

The hydrogels described herein are useful in medical and scientificapplications, wherein the materials comprising nanofibrillar celluloseare in contact with living matter. The products containing nanofibrillarcellulose as described herein are highly biocompatible with the livingmatter and provide several advantageous effects. Without binding to anyspecific theory, it is believed that a hydrogel comprising veryhydrophilic nanofibrillar cellulose having a very high specific surfacearea, and thus high water retention ability, when applied against cellsor tissue, provides favourable moist environment between the cells ortissue and the hydrogel comprising nanofibrillar cellulose. The highamount of free hydroxyl groups in the nanofibrillar cellulose formshydrogen bonds between the nanofibrillar cellulose and water moleculesand enables gel formation and the high water retention ability of thenanofibrillar cellulose. Because of the high amount of water in thenanofibrillar cellulose hydrogel, only water is supposed to be incontact with cells or tissue, and which also enables migration of fluidsand/or agents.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments will be explained in the following with reference to theappended drawings, where

FIG. 1 shows day 5 Hep G2 cell spheroids cultivated in 0.8% GrowDex®hydrogel imaged after 24 hours of trehalose incubation prior to freezingphase. Images (A), (B) and (C) display typical spheroids with a diameterbetween 100 and 120 μm. (D) shows a larger spheroid with a diameter over160 μm.

FIG. 2 shows sterilization of liquid nitrogen. (A) Contaminated liquidnitrogen is poured into a clean bucket. (B) 70% Ethanol is used to spraythe whole system and it is sterilized in a laminar with UV-C light. (C)Inside the laminar, 15 ml tubes are installed to the rack without caps.(D) The tubes are filled with approximately 5 ml of sterilized liquidnitrogen while the tubes are still submerged in liquid nitrogen.

FIG. 3 shows freezing of the cell hydrogels and igniting thefreeze-drying. (A) Rapid freezing by pipetting small droplets ofcell-hydrogel mixtures directly into liquid nitrogen. (B) Transportationof the samples in liquid nitrogen to the freeze-drier and tubes stillcontaining liquid nitrogen once the vacuum is ignited.

FIG. 4 shows (A) A vitrified 200 μl cell sample in frozen state afterrapid freezing. (B) The sample after freeze-drying in dry state.

FIG. 5 shows an optimized freeze-drying cycle for Hep G2 spheroid cellsystems in GrowDex® in 96-wells (200 μl per well) for EPSILON 1-6D byCHRIST resulting in dry samples with minimum time consumed.

FIG. 6 shows (A) A cell flask containing a 96-well plate with cellsamples. (B) A 15 ml tube sealed with sterile Corning® breathablesealing tape.

FIG. 7 shows a standard line drawn from the optimization results ofalamarBlue® for Hep G2 cells alamarBlue® with excitation at 560 nm andemission at 590 nm.

FIG. 8 shows recreated cell hydrogel systems after freeze-drying. (A)extra-large cell spheroids survived the freeze-drying without detachmentof cells. (B) different sized cell spheroids after rehydration. (C)Smaller spheroids after rehydration. (D) A cell spheroid with no changesin the morphology after rehydration.

FIG. 9 shows how a 3D cell spheroid has started to attach itself on a 2Dsurface of a cell culture flask after rehydration and 4 hours afterreseeding.

FIG. 10 shows an overlay image (C) of Live/Dead stained cell spheroidafter rehydration and double staining containing both viable (A) anddead cells (B).

FIG. 11 shows dual stained cells of 2D and 3D cell samples afterfreeze-drying and rehydration. (A) All cells that did not have theprotective effect of GrowDex®, were found dead. (B) Viabilities of15-35% were estimated in samples with GrowDex®.

FIG. 12 shows a cell spheroid after rehydration. Dual staining revealsenzyme activity and cells with lost membrane integrity, while some cellshave both; partially lost membrane integrity and remaining enzymeactivity.

FIG. 13 shows freeze-dried Hep G2 cells and cell spheroids encapsulatedin GrowDex® aerogel. (A), (B), (C) and (D) Spheroids of typical size andshape. (E) A tiny cell spheroid. (F) A single Hep G2 cell.

DETAILED DESCRIPTION

Freeze-drying, also known as lyophilization, is a physical process,where water is removed controllably from a frozen substance bysublimation. It is a sensitive drying method when compared to otherdrying methods, such as oven drying and spray drying, and thereforeespecially suitable for drying proteins and other biological compounds.

Freeze-drying is a multistage operation, in which the success of eachstep is critical for the outcome. In freeze-drying an active substanceis dried that needs to keep its prime properties, such as the correct 3Dstructure in the case of proteins. The process includes a surrounding“medium” which refers to the added bulking agents, such as cryo- andlyoprotectors, stabilizers, emulsifiers and antioxidants. The process ingeneral comprises seven phases, of which in the first one the substanceis prepared by adding the required bulking agents. Next, in the secondphase, the substance is frozen close to its Tg′ point (lower transitiontemperature), which is unique to each mixture. Then in the third step,the substance is dried for an extended period of time in a vacuum andany frozen water is removed through sublimation. This phase is known asthe primary drying phase, or sublimation phase. Under normal conditions,the drying would happen as a phase transition from liquid to gas phase.However, in freeze-drying the temperature is decreased until thesubstance is frozen, and then the pressure is decreased in thesublimation step. A process of shifting takes place through the solidphase around the triple point of water.

Next, in the fourth phase of freeze-drying, any non-frozen bound wateris extracted by increasing the temperature while decreasing thepressure. This step is known as the secondary drying or desorptionphase. When the substance is dry, it has to be removed from thefreeze-drying machinery in the fifth step; conditioning and storage. Inthis phase, the substance easily gets damaged by water, light, oxygenand contaminants. In the sixth step of freeze-drying, the samples areplaced in the ultimate storage. The required storage parameters shouldbe optimized for each substance individually. Lastly, at the seventh,reconstitution phase, the sample is rehydrated in order to recreate theoriginal system for use. If the developed freeze-drying cycle issuccessful, the rehydrated substance should carry the same or nearly thesame properties as the original substance.

The present application discloses the use of nanofibrillar cellulosehydrogel as a lyoprotective matrix in freeze-drying process.Nanofibrillar cellulose matrix was found to be advantageous whenfreeze-drying cells, especially eukaryotic cells, such as mammaliancells. Naturally all kinds of cells may be freeze-dried using themethods disclosed herein, such as prokaryotic cells, as well asextracellular vesicles, such as microvesicles. Cryoprotectants and/orlyoprotectants may be used in the process.

It was found out that the nanofibrillar cellulose used as a matrix actedas a lyoprotectant, which effect could be enhanced by using other cryo-or lyoprotective agents, as disclosed herein. Therefore a synergisticeffect of enhanced cryo- and lyoprotection was obtained.

The present disclosure provides hydrogels comprising nanofibrillarcellulose, which may be also called as nanofibrillar cellulosehydrogels. The hydrogels may be provided as products, which may containalso other substances or other elements, such as reinforcing materials,covering materials, active agents, salts or the like. The hydrogels maybe also provided or called as medical hydrogels or medical products.

The present application provides a method for freeze-drying cells in ahydrogel comprising nanofibrillar cellulose, the method comprising

-   -   providing a hydrogel comprising nanofibrillar cellulose,    -   providing cells,    -   combining the cells and the hydrogel comprising nanofibrillar        cellulose to form a cell system. The cells may be provided in        aqueous medium, such as cell culture medium. Aqueous medium may        also be added to the cell system. When extracellular vesicles        are concerned, the formed system may be called as a vesicle        system or extracellular vesicle system. The procedure with the        vesicles is otherwise similar to the procedure with cells, and        similar products are obtained, only with vesicles instead of        cells. The method and the obtained product may also include both        the cells and the vesicles.

In one embodiment the method comprises culturing the cells in the cellsystem. In such case a cell culture is formed. In many cases the cellsystem may be also called as cell culture.

Initially the cells may be pre-cultured in a separate culture, andrecovered and transferred into a new medium, which may be similar ordifferent than the culture medium. A cell suspension is obtained. This,or another cell suspension, may be combined and/or mixed with thenanofibrillar cellulose, such as a hydrogel comprising nanofibrillarcellulose, to obtain or form a cell system. If cells are cultured in thecell system a cell culture is formed. The cell culture may be 2D cultureor a 3D culture. In one embodiment the cell culture is a 3D cellculture. The cells may be cultured or incubated for a period of time. Inone embodiment the method comprises providing one or more cryoprotectiveand/or lyoprotective agent(s), for example in a culture medium, andadding the one or more cryoprotective and/or lyoprotective agent(s) tothe cell system or to the cell culture. For example the cell culturemedium may be changed into medium containing one or more cryoprotectiveand/or lyoprotective agent(s). Alternatively, or in addition, the one ormore cryoprotective and/or lyoprotective agent(s) may be added to thecell culture medium in the cell culture. In one example trehalose isincluded in the culture medium first and the cells are incubated withtrehalose for a period of time, for example for about 12-24 hours, forexample with 20-100 mM trehalose medium, before any other cryoprotectiveand/or lyoprotective agent(s) is/are added. Finally the method comprisesfreeze drying the cell culture to obtain dried cells in a hydrogelcomprising nanofibrillar cellulose.

One embodiment provides a hydrogel comprising nanofibrillar celluloseand one or more cryoprotective and/or lyoprotective agent(s). Such ahydrogel may be provided for combining with the cells as described inprevious.

The cells may be prokaryotic cells, such as bacterial cells, or they maybe eukaryotic cells. Eukaryotic cells may be plant cells, yeast cells oranimal cells. Examples of eukaryotic cells include transplantable cells,such as stem cells, for example omnipotent, pluripotent, multipotent,oligopotent or unipotent cells. In case of human embryonic stem cellsthe cells may be from a deposited cell line or made from unfertilizedeggs, i.e. “parthenote” eggs or from parthenogenetically activated ovum,so that no human embryos are destroyed. The cells may be cultured in thehydrogel, and they may be also freeze-dried in it. The cells can bemaintained and proliferated on or in the hydrogel without animal orhuman based chemicals originating outside the cells. The cells may beevenly dispersed on or in the hydrogel.

Examples of cells include stem cells, undifferentiated cells, precursorcells, as well as fully differentiated cells and combinations thereof.In some examples the cells comprise cell types selected from the groupconsisting of keratocytes, keratinocytes, fibroblast cells, epithelialcells and combinations thereof. In some examples the cells are selectedfrom the group consisting of stem cells, progenitor cells, precursorcells, connective tissue cells, epithelial cells, muscle cells, neuronalcells, endothelial cells, fibroblasts, keratinocytes, smooth musclecells, stromal cells, mesenchymal cells, immune system cells,hematopoietic cells, dendritic cells, hair follicle cells andcombinations thereof. The cells may be tumor or cancer cells,genetically modified cells, such as transgenic cells, cisgenic cells orknock-out cells, or pathogenic cells. Such cells may be used for examplefor drug research.

In one embodiment the cells are eukaryotic cells, such as mammaliancells. Examples of mammalian cells include human cells, mouse cells, ratcells, rabbit cells, monkey cells, pig cells, bovine cells, chickencells and the like. It is to be noted that even though the advantages ofthe present method are best demonstrated for freeze-drying mammaliancells, the method may be also used for freeze-drying other cells, suchas non-mammalian eukaryotic cells, yeast cells, or prokaryotic cells.

When cells are grown in 3D culture in nanofibrillar cellulose hydrogels,the cells grow as spheroids and may communicate with each other. Such acell colony may be considered as a tissue. According to one definition atissue is an ensemble of similar cells from the same origin thattogether carry out a specific function. Therefore the present method maybe also applied for freeze-drying tissue. It is possible to provide afreeze-dried product comprising a desired cell type, which may be usedinstead of a real tissue for scientific research, for example for drugtesting. A freeze-dried 3D cell culture or tissue culture obtained withthe method disclosed herein is able to continue growth shortly afterrehydration.

A freeze-dried hydrogel may be called as an aerogel, more particularly afreeze-dried aerogel. According to one definition, an aerogel is aporous ultralight material derived from a gel, in which the liquidcomponent of the gel has been replaced with a gas. Despite their name,aerogels are solid, rigid, and dry materials that do not resemble a gelin their physical properties: the name comes from the fact that they aremade from gels.

As the freeze-drying method was found to preserve extracellularvesicles, such as exosomes or microvesicles, it is possible tofreeze-dry extracellular vesicles with the method to obtain driedextracellular vesicles in a hydrogel comprising nanofibrillar cellulose.Such extracellular vesicles, especially microvesicles, may be used in amethod for targeting therapeutical agent(s) to target cells.

Exosomes are cell-derived vesicles that are present in most eukaryoticfluids, including blood, urine, and cultured medium of cell cultures.The diameter of exosomes may be in the range of 30-100 nm. Exosomes maybe released from the cell when multivesicular bodies fuse with theplasma membrane or they are released directly from the plasma membrane.Exosomes contain various molecular constituents of their cell of origin,including proteins, RNA, lipids and metabolites. Although the exosomalprotein composition varies with the cell and tissue of origin, mostexosomes contain an evolutionarily-conserved common set of proteinmolecules. The protein content of a single exosome, given certainassumptions of protein size and configuration, and packing parameters,can be about 20 000 molecules. Exosomes can transfer molecules from onecell to another via membrane vesicle trafficking, thereby influencingthe immune system, such as dendritic cells and B cells, and may play afunctional role in mediating adaptive immune responses to pathogens andtumors.

Exosomes may be used as highly effective drug carriers. Composed ofcellular membranes with multiple adhesive proteins on their surface,exosomes are known to specialize in cell-cell communications and providean exclusive approach for the delivery of various therapeutic agents totarget cells, such as anti-cancer drugs.

Microvesicles are one type of extracellular vesicles. They are circularfragments of plasma membrane having a diameter in the range of 100-1000nm. Microvesicles play a role in intercellular communication and cantransport mRNA, miRNA, and proteins between cells. Microvesicles act inthe process of a remarkable anti-tumor reversal effect in cancer, tumorimmune suppression, metastasis, tumor-stroma interactions andangiogenesis along with having a primary role in tissue regeneration.Microvesicles reflect the antigenic content of the cells from which theyoriginate. Different cells can release microvesicles from the plasmamembrane, such as megakaryocytes, blood platelets, monocytes,neutrophils, tumor cells and placenta.

A dried hydrogel comprising nanofibrillar cellulose, cells andoptionally one or more cryoprotective and/or lyoprotective agent(s) isformed in the method. The hydrogel used or formed in the method may be amedical hydrogel.

The term “medical” refers to a product or use wherein the product, i.e.a product comprising the hydrogel of the embodiments, is used or issuitable for medical purposes or for scientific purposes, for examplefor research. A medical product may be sterilized, or it isserializable, for example by using temperature, pressure, moisture,chemicals, radiation or a combination thereof. The product, preferablythe hydrogel without cells, may be for example autoclaved, or othermethods using high temperature may be used, in which cases the productshould tolerate high temperatures over 100° C., for example at least121° C. or 134° C. In one example the product is autoclaved at 121° C.for 15 minutes. It is also desired that a medical product is pyrogenfree and it does not contain undesired protein residues or the like. Amedical product is preferably non-toxic to the target. Also UVsterilization may be used.

The nanofibrillar cellulose (NFC) hydrogel of the embodiments, such asanionic NFC hydrogel, is able to controllably release active agents,such as agents secreted by the cells, or therapeutic agents, for examplepharmaceutical ingredients, as a function of time, especially when thetemperature and pH are constant. It was found out that NFC hydrogel canbe freeze-dried with the specific excipients and still be re-gelled.Anionic hydrogels are preferred for many applications. For exampleanionically modified nanofibrillar cellulose does not precipitate easilyunlike the other grades. The aniconic grade is also especially suitablefor releasing certain active agents.

In the tests a commercial product manufactured by UPM-Kymmene Oyj calledGrowDex® was used. GrowDex® is a sterile nanofibrillated cellulosehydrogel product manufactured from birch pulp. The native GrowDex®hydrogel is highly viscous, but its viscosity can be adjusted torequired conditions by throughout mixing with water-based liquids, suchas cell media. If the fiber concentration is adjusted below 1.6%, thehydrogel can be dispensed by pipetting while it maintains relevantlyhigh water retention. The hydrogel tolerates salts, changes intemperature and pH and retains its properties at least in the pH rangeof 6-8. However, magnesium and calcium can crosslink NFC fibers andshould therefore be avoided in cell media and other additives forunwanted crosslinking. The ultrastructure of the NFC hydrogel resemblesextracellular matrix (ECM). GrowDex® hydrogel is non-autofluorescent,and therefore compatible with cell staining and imagining. In case ofstains that adhere to NFC fibers, the GrowDex® hydrogel structure can beenzymatically dissembled by addition of cellulase enzyme priorimagining.

In this specification, percentage values, unless specifically indicatedotherwise, are based on weight (w/w). If any numerical ranges areprovided, the ranges include also the upper and lower values.

Nanofibrillar Cellulose

The nanofibrillar cellulose is prepared generally from cellulose rawmaterial of plant origin. The raw material may be based on any plantmaterial that contains cellulose. The raw material may also be derivedfrom certain bacterial fermentation processes. The nanofibrillarcellulose is preferably made of plant material. In one example thefibrils are obtained from non-parenchymal plant material. In such casethe fibrils may be obtained from secondary cell walls. One abundantsource of such cellulose fibrils is wood fibres. The nanofibrillarcellulose is manufactured by homogenizing wood-derived fibrous rawmaterial, which may be chemical pulp. Cellulose fibers are disintegratedto produce fibrils which have the diameter of only some nanometers,which is 50 nm at the most, for example in the range of 1-50 μm, andgives a dispersion of fibrils in water. The fibrils may be reduced tosize where the diameter of most of the fibrils is in the range of only2-20 nm. The fibrils originating from secondary cell walls areessentially crystalline with degree of crystallinity of at least 55%.Such fibrils may have different properties than fibrils originated fromprimary cell walls, for example the dewatering of fibrils originatingfrom secondary cell walls may be more challenging. In general in thecellulose sources from primary cell walls, such as sugar beet, potatotuber and banana rachis, the microfibrils are easier to liberate fromthe fibre matrix than fibrils from wood, and the disintegration requiresless energy. However, these materials are still somewhat heterogeneousand consist of large fibril bundles.

In one embodiment the plant material is wood. Wood may be from softwoodtree such as spruce, pine, fir, larch, douglas-fir or hemlock, or fromhardwood tree such as birch, aspen, poplar, alder, eucalyptus, oak,beech or acacia, or from a mixture of softwoods and hardwoods. In oneembodiment the nanofibrillar cellulose is obtained from wood pulp. Inone embodiment the nanofibrillar cellulose is obtained from hardwoodpulp. In one example the hardwood is birch. In one embodiment thenanofibrillar cellulose is obtained from softwood pulp. In oneembodiment said wood pulp is chemical pulp.

As used herein, the term “nanofibrillar cellulose” refers to cellulosefibrils or fibril bundles separated from cellulose-based fiber rawmaterial. These fibrils are characterized by a high aspect ratio(length/diameter): their length may exceed 1 μm, whereas the diametertypically remains smaller than 200 nm. The smallest fibrils are in thescale of so-called elementary fibrils, the diameter being typically inthe range of 2-12 nm. The dimensions and size distribution of thefibrils depend on the refining method and efficiency. Nanofibrillarcellulose may be characterized as a cellulose-based material, in whichthe median length of particles (fibrils or fibril bundles) is notgreater than 50 μm, for example in the range of 1-50 μm, and theparticle diameter is smaller than 1 μm, suitably in the range of 2-500nm. In case of native nanofibrillar cellulose, in one embodiment theaverage diameter of a fibril is in the range of 5-100 nm, for example inthe range of 10-50 nm. Nanofibrillar cellulose is characterized by alarge specific surface area and a strong ability to form hydrogen bonds.In water dispersion, the nanofibrillar cellulose typically appears aseither light or turbid gel-like material. Depending on the fiber rawmaterial, nanofibrillar cellulose may also contain small amounts ofother wood components, such as hemicellulose or lignin. The amount isdependent on the plant source. Often used parallel names fornanofibrillar cellulose include nanofibrillated cellulose (NFC) andnanocellulose.

In general cellulose nanomaterials may be divided into categoriesaccording to TAPPI W13021, which provides standard terms for cellulosenanomaterials. Two main categories are “Nano objects” and “Nanostructured materials”. Nanostructured materials include “Cellulosemicrocrystals” (sometimes called as CMC) having a width of 10-12 μm andL/D<2, and “Cellulose microfibrils” having a width of 10-100 nm and alength of 0.5-50 μm. Nano objects include “Cellulose nanofibers”, whichcan be divided into “Cellulose nanocrystals” (CNC) having a width of3-10 nm and L/D>5, and “Cellulose nanofibrils” (CNF or NFC), having awidth of 5-30 nm and L/D>50.

Different grades of nanofibrillar cellulose may be categorized based onthree main properties: (i) size distribution, length and diameter (ii)chemical composition, and (iii) rheological properties. To fullydescribe a grade, the properties may be used in parallel. Examples ofdifferent grades include native (or non-modified) NFC, oxidized NFC(high viscosity), oxidized NFC (low viscosity), carboxymethylated NFCand cationized NFC. Within these main grades, also sub-grades exist, forexample: extremely well fibrillated vs. moderately fibrillated, highdegree of substitution vs. low, low viscosity vs. high viscosity etc.The fibrillation technique and the chemical pre-modification have aninfluence on the fibril size distribution. Typically, non-ionic gradeshave wider fibril diameter (for example in the range of 10-100 nm, or10-50 nm) while the chemically modified grades are a lot thinner (forexample in the range of 2-20 nm). Distribution is also narrower for themodified grades. Certain modifications, especially TEMPO-oxidation,yield shorter fibrils.

Depending on the raw material source, e.g. hardwood (HW) vs. softwood(SW) pulp, different polysaccharide composition exists in the finalnanofibrillar cellulose product. Commonly, the non-ionic grades areprepared from bleached birch pulp, which yields high xylene content (25%by weight). Modified grades are prepared either from HW or SW pulps. Inthose modified grades, the hemicelluloses are also modified togetherwith the cellulose domain. Most probably, the modification is nothomogeneous, i.e. some parts are more modified than others. Thus,detailed chemical analysis is not possible—the modified products arealways complicated mixtures of different polysaccharide structures.

In an aqueous environment, a dispersion of cellulose nanofibers forms aviscoelastic hydrogel network. The gel is formed at relatively lowconcentrations of, for example, 0.05-0.2% (w/w) by dispersed andhydrated entangled fibrils. The viscoelasticity of the NFC hydrogel maybe characterized, for example, with dynamic oscillatory rheologicalmeasurements.

The nanofibrillar cellulose hydrogels exhibit characteristic rheologicalproperties. For example they are shear-thinning or pseudoplasticmaterials, which means that their viscosity depends on the speed (orforce) by which the material is deformed. When measuring the viscosityin a rotational rheometer, the shear-thinning behavior is seen as adecrease in viscosity with increasing shear rate. The hydrogels showplastic behavior, which means that a certain shear stress (force) isrequired before the material starts to flow readily. This critical shearstress is often called the yield stress. The yield stress can bedetermined from a steady state flow curve measured with a stresscontrolled rheometer. When the viscosity is plotted as function ofapplied shear stress, a dramatic decrease in viscosity is seen afterexceeding the critical shear stress. The zero shear viscosity and theyield stress are the most important rheological parameters to describethe suspending power of the materials. These two parameters separate thedifferent grades quite clearly and thus enable classification of thegrades.

The dimensions of the fibrils or fibril bundles are dependent on the rawmaterial and the disintegration method. Mechanical disintegration of thecellulose raw material may be carried out with any suitable equipmentsuch as a refiner, grinder, disperser, homogenizer, colloider, frictiongrinder, pin mill, rotor-rotor dispergator, ultrasound sonicator,fluidizer such as microfluidizer, macrofluidizer or fluidizer-typehomogenizer. The disintegration treatment is performed at conditionswherein water is sufficiently present to prevent the formation of bondsbetween the fibers.

In one example the disintegration is carried out by using a disperserhaving at least one rotor, blade or similar moving mechanical member,such as a rotor-rotor dispergator, which has at least two rotors. In adisperser the fiber material in dispersion is repeatedly impacted byblades or ribs of rotors striking it from opposite directions when theblades rotate at the rotating speed and at the peripheral speeddetermined by the radius (distance to the rotation axis) in oppositedirections. Because the fiber material is transferred outwards in theradial direction, it crashes onto the wide surfaces of the blades, i.e.ribs, coming one after the other at a high peripheral speed fromopposite directions; in other words, it receives several successiveimpacts from opposite directions. Also, at the edges of the widesurfaces of the blades, i.e. ribs, which edges form a blade gap with theopposite edge of the next rotor blade, shear forces occur, whichcontribute to the disintegration of the fibers and detachment offibrils. The impact frequency is determined by the rotation speed of therotors, the number of the rotors, the number of blades in each rotor,and the flow rate of the dispersion through the device.

In a rotor-rotor dispergator the fiber material is introduced throughcounter-rotating rotors, outwards in the radial direction with respectto the axis of rotation of the rotors in such a way that the material isrepeatedly subjected to shear and impact forces by the effect of thedifferent counter-rotating rotors, whereby it is simultaneouslyfibrillated. One example of a rotor-rotor dispergator is an Atrexdevice.

Another example of a device suitable for disintegrating is a pin mill,such as a multi-peripheral pin mill. One example of such device, asdescribed in U.S. Pat. No. 6,202,946 B1, includes a housing and in it afirst rotor equipped with collision surfaces; a second rotor concentricwith the first rotor and equipped with collision surfaces, the secondrotor being arranged to rotate in a direction opposite to the firstrotor; or a stator concentric with the first rotor and equipped withcollision surfaces. The device includes a feed orifice in the housingand opening to the center of the rotors or the rotor and stator, and adischarge orifice on the housing wall and opening to the periphery ofthe outermost rotor or stator.

In one embodiment the disintegrating is carried out by using ahomogenizer. In a homogenizer the fiber material is subjected tohomogenization by an effect of pressure. The homogenization of the fibermaterial dispersion to nanofibrillar cellulose is caused by forcedthrough-flow of the dispersion, which disintegrates the material tofibrils. The fiber material dispersion is passed at a given pressurethrough a narrow through-flow gap where an increase in the linearvelocity of the dispersion causes shearing and impact forces on thedispersion, resulting in the removal of fibrils from the fiber material.The fiber fragments are disintegrated into fibrils in the fibrillatingstep.

As used herein, the term “fibrillation” generally refers todisintegrating fiber material mechanically by work applied to theparticles, where cellulose fibrils are detached from the fibers or fiberfragments. The work may be based on various effects, like grinding,crushing or shearing, or a combination of these, or anothercorresponding action that reduces the particle size. The energy taken bythe refining work is normally expressed in terms of energy per processedraw material quantity, in units of e.g. kWh/kg, MWh/ton, or unitsproportional to these. The expressions “disintegration” or“disintegration treatment” may be used interchangeably with“fibrillation”.

The fiber material dispersion that is subjected to fibrillation is amixture of fiber material and water, also herein called “pulp”. Thefiber material dispersion may refer generally to whole fibers, parts(fragments) separated from them, fibril bundles, or fibrils mixed withwater, and typically the aqueous fiber material dispersion is a mixtureof such elements, in which the ratios between the components aredependent on the degree of processing or on the treatment stage, forexample number of runs or “passes” through the treatment of the samebatch of fiber material.

One way to characterize the nanofibrillar cellulose is to use theviscosity of an aqueous solution containing said nanofibrillarcellulose. The viscosity may be, for example, Brookfield viscosity orzero shear viscosity. The specific viscosity, as described herein,distinguishes nanofibrillar cellulose from non-nanofibrillar cellulose.

In one example the apparent viscosity of the nanofibrillar cellulose ismeasured with a Brookfield viscometer (Brookfield viscosity) or anothercorresponding apparatus. Suitably a vane spindle (number 73) is used.There are several commercial Brookfield viscometers available formeasuring apparent viscosity, which all are based on the same principle.Suitably RVDV spring (Brookfield RVDV-III) is used in the apparatus. Asample of the nanofibrillar cellulose is diluted to a concentration of0.8% by weight in water and mixed for 10 min. The diluted sample mass isadded to a 250 ml beaker and the temperature is adjusted to 20° C.±1°C., heated if necessary and mixed. A low rotational speed 10 rpm isused.

The nanofibrillar cellulose provided as a starting material in themethod may be characterized by the viscosity it provides in a watersolution. The viscosity describes, for example, the fibrillation degreeof the nanofibrillar cellulose. In one embodiment the nanofibrillarcellulose when dispersed in water provides a Brookfield viscosity of atleast 2000 mPa·s, such as at least 3000 mPa·s, measured at a consistencyof 0.8% (w/w) and at 10 rpm. In one embodiment the nanofibrillarcellulose, when dispersed in water, provides a Brookfield viscosity ofat least 10000 mPa·s measured at a consistency of 0.8% (w/w) and at 10rpm. In one embodiment the nanofibrillar cellulose, when dispersed inwater, provides a Brookfield viscosity of at least 15000 mPa·s measuredat a consistency of 0.8% (w/w) and at 10 rpm. Examples of Brookfieldviscosity ranges of said nanofibrillar cellulose when dispersed in waterinclude 2000-20000 mPa·s, 3000-20000 mPa·s, 10000-20000 mPa·s,15000-20000 mPa·s, 2000-25000 mPa·s, 3000-25000 mPa·s, 10000-25000mPa·s, 15000-25000 mPa·s, 2000-30000 mPa·s, 3000-30000 mPa·s,10000-30000 mPa·s, and 15000-30000 mPa·s, measured at a consistency of0.8% (w/w) and at 10 rpm.

In one embodiment the nanofibrillar cellulose comprises non-modifiednanofibrillar cellulose. In one embodiment the nanofibrillar celluloseis non-modified nanofibrillar cellulose. It was found out that thedrainage of non-modified nanofibrillar cellulose was significantlyfaster than with for example anionic grade. Non-modified nanofibrillarcellulose generally has a Brookfield viscosity in the range of2000-10000 mPa·s, measured at a consistency of 0.8% (w/w) and at 10 rpm.

The disintegrated fibrous cellulosic raw material may be modifiedfibrous raw material. Modified fibrous raw material means raw materialwhere the fibers are affected by the treatment so that cellulosenanofibrils are more easily detachable from the fibers. The modificationis usually performed to fibrous cellulosic raw material which exists asa suspension in a liquid, that is, pulp.

The modification treatment to the fibers may be chemical or physical. Inchemical modification the chemical structure of cellulose molecule ischanged by chemical reaction (“derivatization” of cellulose), preferablyso that the length of the cellulose molecule is not affected butfunctional groups are added to β-D-glucopyranose units of the polymer.The chemical modification of cellulose takes place at a certainconversion degree, which is dependent on the dosage of reactants and thereaction conditions, and as a rule it is not complete so that thecellulose will stay in solid form as fibrils and does not dissolve inwater. In physical modification anionic, cationic, or non-ionicsubstances or any combination of these are physically adsorbed oncellulose surface. The modification treatment may also be enzymatic.

The cellulose in the fibers may be especially ionically charged afterthe modification, because the ionic charge of the cellulose weakens theinternal bonds of the fibers and will later facilitate thedisintegration to nanofibrillar cellulose. The ionic charge may beachieved by chemical or physical modification of the cellulose. Thefibers may have higher anionic or cationic charge after the modificationcompared with the starting raw material. Most commonly used chemicalmodification methods for making an anionic charge are oxidation, wherehydroxyl groups are oxidized to aldehydes and carboxyl groups,sulphonization and carboxymethylation. A cationic charge in turn may becreated chemically by cationization by attaching a cationic group to thecellulose, such as quaternary ammonium group.

In one embodiment the nanofibrillar cellulose comprises chemicallymodified nanofibrillar cellulose, such as anionically modifiednanofibrillar cellulose or cationically modified nanofibrillarcellulose. In one embodiment the nanofibrillar cellulose is anionicallymodified nanofibrillar cellulose. In one embodiment the nanofibrillarcellulose is cationically modified nanofibrillar cellulose. In oneembodiment the anionically modified nanofibrillar cellulose is oxidizednanofibrillar cellulose. In one embodiment the anionically modifiednanofibrillar cellulose is sulphonized nanofibrillar cellulose. In oneembodiment the anionically modified nanofibrillar cellulose iscarboxymethylated nanofibrillar cellulose. Chemically modifiednanofibrillar celluloses may be used to affect to the release profile ofcertain active agents. For example anionic grades may be used to releasecationically charged molecules to obtain a prolonged release rate, orvice versa.

The cellulose may be oxidized. In the oxidation of cellulose, theprimary hydroxyl groups of cellulose may be oxidized catalytically by aheterocyclic nitroxyl compound, for example2,2,6,6-tetramethylpiperidinyl-1-oxy free radical, generally called“TEMPO”. The primary hydroxyl groups (C6-hydroxyl groups) of thecellulosic β-D-glucopyranose units are selectively oxidized tocarboxylic groups. Some aldehyde groups are also formed from the primaryhydroxyl groups. Regarding the finding that low degree of oxidation doesnot allow efficient enough fibrillation and higher degree of oxidationinflicts degradation of cellulose after mechanical disruptive treatment,the cellulose may be oxidized to a level having a carboxylic acidcontent in the oxidized cellulose in the range of 0.6-1.4 mmol COOH/gpulp, or 0.8-1.2 mmol COOH/g pulp, preferably to 1.0-1.2 mmol COOH/gpulp, determined by conductometric titration. When the fibers ofoxidized cellulose so obtained are disintegrated in water, they givestable transparent dispersion of individualized cellulose fibrils, whichmay be, for example, of 3-5 nm in width. With oxidized pulp as thestarting medium, it is possible to obtain nanofibrillar cellulose whereBrookfield viscosity measured at a consistency of 0.8% (w/w) is at least10000 mPa·s, for example in the range of 10000-30000 mPa·s.

Whenever the catalyst “TEMPO” is mentioned in this disclosure, it isevident that all measures and operations where “TEMPO” is involved applyequally and analogously to any derivative of TEMPO or any heterocyclicnitroxyl radical capable of catalyzing selectively the oxidation of thehydroxyl groups of C6 carbon in cellulose.

In one embodiment such chemically modified nanofibrillar cellulose, whendispersed in water, provides a Brookfield viscosity of at least 10000mPa·s measured at a consistency of 0.8% (w/w) and at 10 rpm. In oneembodiment such chemically modified nanofibrillar cellulose, whendispersed in water, provides a Brookfield viscosity of at least 15000mPa·s measured at a consistency of 0.8% (w/w) and at 10 rpm. In oneembodiment such chemically modified nanofibrillar cellulose, whendispersed in water, provides a Brookfield viscosity of at least 18000mPa·s measured at a consistency of 0.8% (w/w) and at 10 rpm. Examples ofanionic nanofibrillar celluloses used have a Brookfield viscosity in therange of 13000-15000 mPa·s or 18000-20000 mPa·s, or even up to 25000mPa·s, depending on the degree of fibrillation.

In one embodiment the nanofibrillar cellulose is TEMPO oxidizednanofibrillar cellulose. It provides high viscosity at lowconcentrations, for example a Brookfield viscosity of at least 20000mPa·s, even at least 25000 mPa·s, measured at a consistency of 0.8%(w/w) and at 10 rpm. In one example the Brookfield viscosity of TEMPOoxidized nanofibrillar cellulose is in the range of 20000-30000 mPa·s,such as 25000-30000 mPa·s, measured at a consistency of 0.8% (w/w) andat 10 rpm.

In one embodiment the nanofibrillar cellulose comprises chemicallyunmodified nanofibrillar cellulose. In one embodiment such chemicallyunmodified nanofibrillar cellulose, when dispersed in water, provides aBrookfield viscosity of at least 2000 mPa·s, or at least 3000 mPa·s,measured at a consistency of 0.8% (w/w) and at 10 rpm.

The nanofibrillar cellulose may also be characterized by the averagediameter (or width), or by the average diameter together with theviscosity, such as Brookfield viscosity or zero shear viscosity. In oneembodiment said nanofibrillar cellulose has a number average diameter ofa fibril in the range of 1-100 nm. In one embodiment said nanofibrillarcellulose has a number average diameter of a fibril in the range of 1-50nm, such as 5-30 nm. In one embodiment said nanofibrillar cellulose hasa number average diameter of a fibril in the range of 2-15 nm, such asTEMPO oxidized nanofibrillar cellulose.

The diameter of a fibril may be determined with several techniques, suchas by microscopy. Fibril thickness and width distribution may bemeasured by image analysis of the images from a field emission scanningelectron microscope (FE-SEM), a transmission electron microscope (TEM),such as a cryogenic transmission electron microscope (cryo-TEM), or anatomic force microscope (AFM). In general AFM and TEM suit best fornanofibrillar cellulose grades with narrow fibril diameter distribution.

In one example a rheometer viscosity of the nanofibrillar cellulosedispersion is measured at 22° C. with a stress controlled rotationalrheometer (AR-G2, TA Instruments, UK) equipped with a narrow gap vanegeometry (diameter 28 mm, length 42 mm) in a cylindrical sample cuphaving a diameter of 30 mm. After loading the samples to the rheometerthey are allowed to rest for 5 min before the measurement is started.The steady state viscosity is measured with a gradually increasing shearstress (proportional to applied torque) and the shear rate (proportionalto angular velocity) is measured. The reported viscosity (=shearstress/shear rate) at a certain shear stress is recorded after reachinga constant shear rate or after a maximum time of 2 min. The measurementis stopped when a shear rate of 1000 s-1 is exceeded. This method may beused for determining the zero-shear viscosity.

In one example the nanofibrillar cellulose, when dispersed in water,provides a zero shear viscosity (“plateau” of constant viscosity atsmall shearing stresses) in the range of 1000-100000 Pas, such as in therange of 5000-50000 Pas, and a yield stress (shear stress where theshear thinning begins) in the range of 1-50 Pa, such as in the range of3-15 Pa, determined by rotational rheometer at a consistency of 0.5%(w/w) by weight in aqueous medium.

Turbidity is the cloudiness or haziness of a fluid caused by individualparticles (total suspended or dissolved solids) that are generallyinvisible to the naked eye. There are several practical ways ofmeasuring turbidity, the most direct being some measure of attenuation(that is, reduction in strength) of light as it passes through a samplecolumn of water. The alternatively used Jackson Candle method (units:Jackson Turbidity Unit or JTU) is essentially the inverse measure of thelength of a column of water needed to completely obscure a candle flameviewed through it.

Turbidity may be measured quantitatively using optical turbiditymeasuring instruments. There are several commercial turbidometersavailable for measuring turbidity quantitatively. In the present casethe method based on nephelometry is used. The units of turbidity from acalibrated nephelometer are called Nephelometric Turbidity Units (NTU).The measuring apparatus (turbidometer) is calibrated and controlled withstandard calibration samples, followed by measuring of the turbidity ofthe diluted NFC sample.

In one turbidity measurement method, a nanofibrillar cellulose sample isdiluted in water, to a concentration below the gel point of saidnanofibrillar cellulose, and turbidity of the diluted sample ismeasured. Said concentration where the turbidity of the nanofibrillarcellulose samples is measured is 0.1%. HACH P2100 Turbidometer with a 50ml measuring vessel is used for turbidity measurements. The dry matterof the nanofibrillar cellulose sample is determined and 0.5 g of thesample, calculated as dry matter, is loaded in the measuring vessel,which is filled with tap water to 500 g and vigorously mixed by shakingfor about 30 s. Without delay the aqueous mixture is divided into 5measuring vessels, which are inserted in the turbidometer. Threemeasurements on each vessel are carried out. The mean value and standarddeviation are calculated from the obtained results, and the final resultis given as NTU units.

One way to characterize nanofibrillar cellulose is to define both theviscosity and the turbidity. Low turbidity refers to small size of thefibrils, such as small diameter, as small fibrils scatter light poorly.In general as the fibrillation degree increases, the viscosity increasesand at the same time the turbidity decreases. This happens, however,until a certain point. When the fibrillation is further continued, thefibrils finally begin to break and cannot form a strong network anymore. Therefore, after this point, both the turbidity and the viscositybegin to decrease.

In one example the turbidity of anionic nanofibrillar cellulose is lowerthan 90 NTU, for example from 3 to 90 NTU, such as from 5 to 60, forexample 8-40 measured at a consistency of 0.1% (w/w) in aqueous medium,and measured by nephelometry. In one example the turbidity of nativenanofibrillar may be even over 200 NTU, for example from 10 to 220 NTU,such as from 20 to 200, for example 50-200 measured at a consistency of0.1% (w/w) in aqueous medium, and measured by nephelometry. Tocharacterize the nanofibrillar cellulose these ranges may be combinedwith the viscosity ranges of the nanofibrillar cellulose, such asnanofibrillar cellulose which, when dispersed in water, provides aBrookfield viscosity of at least 2000 mPa·s, at least 3000 mPa·s, atleast 5000 mPa·s, such as at least 10000 mPa·s, for example at least15000 mPa·s measured at a consistency of 0.8% (w/w) and at 10 rpm.

The starting material for the preparation process is usuallynanofibrillar cellulose obtained directly from the disintegration ofsome of the above-mentioned fibrous raw material and existing at arelatively low concentration homogeneously distributed in water due tothe disintegration conditions. The starting material may be an aqueousgel at a concentration of 0.2-10%.

In one embodiment the concentration of the nanofibrillar cellulose inthe hydrogel before the freeze-drying is 10% (w/w) or less, or less than10% (w/w), such as in the range of 0.1-10% (w/w), 0.1-5% (w/w), 0.1-2%(w/w), or 0.1-1.0% (w/w). A preferable range useful for cell cultureapplications is 0.1-2% (w/w), 0.1-1% (w/w) or 0.5-1% (w/w). With aconcentration below 0.5% (w/w) many cells do not grow as spheroids in 3Dculture. On the other hand, a concentration of 1.0% (w/w) or more is toohigh for many cells. The same concentrations of the hydrogels may berestored after freeze-drying from the freeze-dried gels. Moreparticularly dried gel is redispersable in water and will give, whenredispersed in water, for example at a dispergation concentration in therange of 0.1-10% (w/w), such as in the ranges disclosed in previous, aviscosity profile that is equal or substantially equal to the viscosityprofile it had originally at the same dispergation concentration.

Cryo- and Lyoprotectants

Cryoprotectants (which may be also called as excipients orcryoprotective agents) contribute to the preservation of the structuresof proteins, liposome bilayers and other substances during freezing ingeneral. Lyoprotectants stabilize these substances during drying,especially freeze-drying. In freeze-drying lyoprotectant may be alsoconsidered as a cryoprotectant, so as used herein the term“cryoprotectant” may also include lyoprotectants. Protective additivescan be generally considered to have two types: (i) amorphous glassforming, and (ii) eutectic crystallizing salts. Examples oflyoprotectants include polyhydroxy compounds such as sugars (mono-, di-,and polysaccharides), trehalose and sucrose as natural lyoprotectantsand polyalcohols, such as glycerol, and their derivatives. Both of thesegroups belong to the type (i). Not all cryoprotectants are suitablecompounds as lyoprotectants, however. For example, in thecryopreservation of cells, DMSO is commonly used as a cryoprotectant asup to 5% (v/v). However, as it is an organic solvent, its usage infreeze-drying is limited due to evaporation, as its freezing point insolutions is relevantly high. Therefore the use of DMSO ascryoprotectant is not desired in the present method.

In one embodiment the method comprises providing one or morecryoprotective and/or lyoprotective agent(s). In one embodiment themethod comprises providing at least one lyoprotective agent, preferablyin addition to the nanofibrillar cellulose hydrogel. In one embodimentthe method comprises providing at least one cryoprotective andlyoprotective agent(s), which may be the same agent, such as trehaloseor PEG, or different agents. One group of cryoprotective agent(s)include alcohols containing at least two hydroxyl groups, such asglycols, for example ethylene glycol, propylene glycol and glycerol. Itwas discovered that useful cryoprotective agent(s) for cells includetrehalose, glycerol and polyethylene glycol (PEG). In one embodiment theone or more cryoprotective and/or lyoprotective agent(s) is selectedfrom trehalose, glycerol, and polyethylene glycol. In one embodiment thecryoprotective and/or lyoprotective agent comprises trehalose. In oneembodiment the cryoprotective and/or lyoprotective agents comprisetrehalose and glycerol. This combination was discovered to provideespecially good cryo- and lyoprotection for cells in a NFC hydrogelmatrix. In one embodiment the cryoprotective and/or lyoprotective agentscomprise trehalose and polyethylene glycol. In one embodiment thecryoprotective and/or lyoprotective agent(s) comprise glycerol andpolyethylene glycol. In one embodiment the cryoprotective and/orlyoprotective agent(s) comprise trehalose, glycerol and polyethyleneglycol. Trehalose, also known as α,α-trehalose;α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside, mycose or tremalose, is anatural alpha-linked disaccharide formed by an α,α-1,1-glucoside bondbetween two α-glucose units. Trehalose is nutritionally equivalent toglucose, because it is rapidly broken down into glucose by the enzymetrehalase. Trehalose may be present as anhydrous or as dihydrate. In oneembodiment the trehalose is D(+)-trehalose dehydrate, which iscompatible with nanofibrillar cellulose. In one example the trehalose isD-(+)-trehalose dehydrate. It may be provided as solid or as dissolvedin an aqueous medium, such as water. Trehalose was found out to act asboth cryoprotective and lyoprotective agent for cells in nanofibrillarcellulose hydrogel.

Glycerol is a simple polyol compound. It is a colorless, odorless,viscous liquid that is sweet-tasting and non-toxic. The glycerolbackbone is found in all lipids known as triglycerides. Glycerol hasthree hydroxyl groups that are responsible for its solubility in waterand its hygroscopic nature. Glycerol was found out to act mostly as acryoprotective agent for the cells in nanofibrillar cellulose hydrogel.

Polyethylene glycol (PEG) is a polyether compound also known aspolyethylene oxide (PEO) or polyoxyethylene (POE), depending on itsmolecular weight. The structure of PEG is commonly expressed asH—(O—CH₂—CH₂)n-OH. In general polyethylene glycols are prepared bypolymerization of ethylene oxide and are commercially available over awide range of molecular weights from 300 g/mol to 10000000 g/mol.Polyethylene glycol is water-soluble and it has a low toxicity.Polyethylene glycol was found out to act as both cryoprotective andlyoprotective agent for cells in nanofibrillar cellulose hydrogel.

In one embodiment the polyethylene glycol has a molecular weight in therange of 100-10000 kDa, such as 1000-10000 kDa. In one embodiment thepolyethylene glycol has a molecular weight in the range of 3000-8000kDa, such as 5000-7000 kDa, for example about 6000 kDa. A “molecularweight” as used in this disclosure may refer to number average molarmass. In general the number average molecular mass of a polymer can bedetermined for example by gel permeation chromatography, viscometry viathe (Mark-Houwink equation), colligative methods such as vapor pressureosmometry, end-group determination or proton NMR.

Preparation of the Freeze Dried Product

The method comprises providing the ingredients nanofibrillar cellulose,in general as an aqueous suspension or hydrogel, cells, and the desiredcryo- and/or lyoprotectant(s). In one embodiment the nanofibrillarcellulose is the only cellulosic material in the aqueous suspension orin the hydrogel. In one embodiment the nanofibrillar cellulose is theonly polymeric gel-forming material in the aqueous suspension or in thehydrogel. In one example the aqueous suspension or the hydrogelcomprises an amount of another fibrous material, such asnon-nanofibrillar cellulose, for example an amount in the dried hydrogel(w/w) of the dry weight of the fibrous material.

The method comprises providing a hydrogel comprising nanofibrillarcellulose. The concentration of the nanofibrillar cellulose in thehydrogel may in the range of 0.1-10% (w/w), 0.1-10% (w/w), 0.1-5% (w/w),0.1-2% (w/w), or 0.1-1.0% (w/w). A preferable range useful for cellculture applications is 0.1-2% (w/w), 0.1-1% (w/w) or 0.5-1% (w/w).

The cells are combined with the hydrogel comprising nanofibrillarcellulose to form a cell system. The cells may be cultured in the cellsystem. In one example the cells are combined with the hydrogelcomprising nanofibrillar cellulose to form a cell culture. A suitablecell culture medium is provided.

The “cell culture medium”, “culture medium”, “medium”, as used hereinfor use with cells, refers to any suitable medium which is adapted to becompatible with cells. Such medium is usually liquid aqueous andcontains auxiliary agents, such as nutrients, salts, vitamins, and/orother agents such as hormones, growth factors and the like. Blood serumor synthetic serum may be included. The medium may be used for culturingthe cells, for suspending the cells, and/or for rehydrating the driedgel/cells. Preferably the medium does not contain calcium or magnesium.

The method comprises providing one or more cryoprotective agent(s), suchas one or more cryoprotective and/or lyoprotective agent(s), for examplein a culture medium, and adding the one or more cryoprotective agent(s)to the cell system or to the cell culture. For example the cell culturemedium may be changed into medium containing one or more cryoprotectiveand/or lyoprotective agent(s). The hydrogel comprising nanofibrillarcellulose may be provided as containing one or more cryoprotectiveand/or lyoprotective agent(s), such as at least trehalose.

In one example the cryoprotective and/or lyoprotective agent(s) comprisetrehalose and glycerol, wherein the concentration of trehalose in theculture medium may be in the range of 0.2-0.5% (w/w) and theconcentration of glycerol in the culture medium may be in the range of0.5-1.5% (w/w), for example about 0.3% (w/w) of trehalose and 1% (w/w)of glycerol. The gel concentration may be in the range of 0.3-1.0%(w/w), such as 0.3-0.5% (w/w).

In case of glycerol and trehalose as cryoprotective and/or lyoprotectiveagents, the ratio of dry nanofibrillar cellulose in the gel to thecryoprotectants (NFC:glycerol:trehalose by weight) may be for exampleabout 9.5:3:1 to 20:3:1. In some examples the ratio of dry nanofibrillarcellulose in the gel to the cryoprotectants (NFC:glycero:trehalose) isin the range of 5-40:2-6:1 by weight, 9-22:2-6:1 by weight, or9.5-20:2-5:1 by weight, or 9-22:2-4:1 by weight, or 9.5-20:2-4:1 byweight, for example about 10:5:1 by weight or about 10:5:1 by weight,about 15:5:1 by weight, about 10:3:1 by weight, about 15:3:1 by weightor about 20:3:1 by weight.

Alternatively, or in addition, the one or more cryoprotective and/orlyoprotective agent(s) may be added to the cell culture medium in thecell culture. The concentrations explained in previous may be obtainedin the medium. In one example trehalose is included in the culturemedium first and the cells are incubated with trehalose for a period oftime, for example for about 12-24 hours, for example with 20-100 mMtrehalose medium, before other cryoprotectants are added.

The obtained mixture, gel, cell system, extracellular vesicle system orcell culture is freeze-dryable, i.e. the freeze-drying process does nothave remarkable effect to the physical properties of the driednanofibrillar gel, the vesicles or the cells. The mixture may be alsocalled as a freeze-dryable hydrogel. One example of such physicalproperty of the hydrogel is a release profile of an agent from thehydrogel, such as small or large molecule(s), for example therapeuticagent(s). It was found out that different molecules having a variety ofmolecular weights, including relatively small organic molecules as wellas larger proteins, could be released from the hydrogel in a controlledway with similar release profile. The useful molecular weight range isvery broad, for example in the tests molecules having a molecular weightin the range of about 170-70 000 g/mol (Daltons) could be releasedcontrollably. However, the molecules with high molecular weight didrelease slower than the molecules with lower molecular weight. Oneexample of such physical property of the cells is the integrity of thecells, such as preservation of cell membranes, cell organelles,microvesicles, and/or other parts of the cells. Another example of thephysical property of cells is the three-dimensional form of the cellcolony.

In one embodiment the method comprises providing one or more therapeuticor other active agent(s) and mixing the agent(s) with the hydrogel,optionally cryoprotective and/or lyoprotective agent(s) and/or thecells, for example adding the agent(s) to the medium. The agent(s) maybe added simultaneously with the one or more cryoprotectant(s), or theagents(s) may be added before or afterwards. Similarly any otheradditives may be provided and mixed with the hydrogel or the medium,such as chemical(s), nutrient(s), cell culturing agent(s) and the like.

After the cell system or the vesicle system has been obtained, it isfreeze-dried to obtain a dried hydrogel, or more particularly anaerogel, comprising nanofibrillar cellulose. Any suitable freeze-dryingmethod may be used. Freeze drying, which may also be called aslyophilisation, is a method which uses rapid cooling to producethermodynamic instability within a system and cause phase separation.The solvent is then removed by sublimation under vacuum leaving behindvoids in the regions it previously occupied. Sublimation refers totransition of a substance directly from the solid to the gas phasewithout passing through the intermediate liquid phase. Sublimation is anendothermic phase transition that occurs at temperatures and pressuresbelow a substance's triple point in its phase diagram.

In one embodiment the freeze drying comprises first lowering thetemperature of the cell system to at least to −30° C., such as at least−40° C., for example to the range of −30-−100° C., or to the range of−40-−100° C., or even to about −200° C. or below, for example when usingliquid nitrogen, and after that applying lowered pressure to removewater from the cell system. In general the cell system should be frozenbefore applying the lowered pressure. In one embodiment the temperatureis increased during applying lowered pressure and after applying thelowest pressure, for example the temperature is increased to about −20°C. or even to about 10° C. The temperature may be increased before thelowered pressure is applied, or it may be increased during applying thelowered pressure.

In one example the freeze drying is carried out by freezing the cellsystem with liquid nitrogen. For example a vial containing the cellsystem is dipped into liquid nitrogen until the cell system is frozen.In another example, which yielded better vitrification in the tests, thecells are pipetted directly into liquid nitrogen. After this the loweredpressure is applied to the cell system to remove water from the cellsystem. The lowered pressure may refer to vacuum required to obtain thesublimation of the water. As the sublimation of the water takes placeunder the triple point, the required vacuum pressure is dependent on theused temperature. The vesicle systems may be freeze-dried using the sameprocedure.

“Drying” as used herein refers in general to dewatering, which terms maybe used interchangeably, wherein water is removed from a hydrogel toobtain dried or dewatered hydrogel. In one embodiment the freeze dryingis continued until the hydrogel has a desired moisture content or thefreeze drying is continued to a minimum moisture content, preferablybelow 20%, or more preferably below 10%, or even below 5%, for exampleto a moisture content in the range of 1-20%, 2-20%, or 2-10% (w/w). Inone embodiment the freeze drying is continued until the hydrogel hasmoisture content in the range of 1-8%, 2-8%, 2-6%, 2-5% or 1-5% (w/w).In general it may be challenging to obtain moisture content below 2%.After the low moisture content has been obtained, the dried product maybe packed into a package in vacuum or in protective gas. This willprevent the dried hydrogel absorbing the ambient moisture, which mightraise the moisture content to a range of for example 4-8%, or 5-7%(w/w). The moisture content may be also called as water content. Theobtained dried hydrogel may be regelled by adding aqueous liquid, suchas culture medium, and suspending the dried product. A regelled orresuspended hydrogel is obtained, which may have the same concentrationand water content as before drying. This hydrogel providescharacteristics which are substantially equal to the characteristics ofthe original hydrogel before drying.

Final freeze-dried hydrogels, more particularly freeze-dried aerogels,comprising nanofibrillar cellulose comprising the cells are obtainedwith the freeze-drying methods of the embodiments disclosed herein. Thefreeze-dried aerogels may be stored in sealed packages or containers.Inert gas may be provided, such as argon. The freeze-dried products maybe stored at lowered temperature, such as in a refrigerator, for exampleat 0-10° C., such as at about +4° C., or even at room temperature.

One embodiment provides a freeze-dried aerogel comprising nanofibrillarcellulose, cells and one or more cryoprotective and/or lyoprotectiveagent(s), wherein the moisture content of the hydrogel is 10% or less(w/w), preferably in the range of 2-10% (w/w), such as 2-8% (w/w), asdiscussed in the previous. The freeze-dried aerogel may further compriseone or more therapeutic or other active agent(s), or other agents, asdiscussed herein. Actually such a product is no longer in a form of agel, which in the case of nanofibrillar cellulose generally has amoisture content over 80% (w/w), or over 90% (w/w), even over 95% (w/w).The freeze-dried (nanofibrillar cellulose) aerogel may be thereforecalled as freeze dried nanofibrillar cellulose.

One embodiment provides the freeze-dried aerogel comprisingnanofibrillar cellulose, wherein the content of the cells in thefreeze-dried aerogel is in the range of 0.1-65% (w/w), such as in therange of 0.1-50% (w/w), such as in the range of 1-25% (w/w), or 1-20%(w/w), 1-10% (w/w), 1-5% (w/w), or 20-65% (w/w), 10-65% (w/w), 5-65%(w/w), 10-50% (w/w), 5-50% (w/w), 5-25% (w/w), 5-20% (w/w), or 5-15%(w/w).

One embodiment provides the freeze-dried aerogel comprisingnanofibrillar cellulose, wherein the content of the extracellularvesicles in the freeze-dried aerogel is in the range of 0.1-65% (w/w),such as in the range of 0.1-50% (w/w), such as in the range of 1-25%(w/w), or 1-20% (w/w), 1-10% (w/w), 1-5% (w/w), or 20-65% (w/w), 10-65%(w/w), 5-65% (w/w), 10-50% (w/w), 5-50% (w/w), 5-25% (w/w), 5-20% (w/w),or 5-15% (w/w).

One embodiment provides the freeze-dried aerogel, wherein the content ofthe one or more therapeutic or other active agent or other chemical inthe freeze-dried hydrogel is in the range of 0.1-65% (w/w), or in therange of 0.1-50% (w/w), such as in the range of 1-25% (w/w), or 1-20%(w/w), 1-10% (w/w), 1-5% (w/w), or 20-65% (w/w), 10-65% (w/w), 5-65%(w/w), 10-50% (w/w), 5-50% (w/w), 5-25% (w/w), 5-20% (w/w), or 5-15%(w/w).

In one embodiment the content of the glycerol is in the range of 1-10%(w/w) in the dried hydrogel, such as in the range of 5-10% (w/w). In oneembodiment the content of the trehalose is in the range of 0.5-8% (w/w)in the dried hydrogel, such as in the range of 3-4% (w/w). In oneembodiment the content of the polyethylene glycol is in the range of1-10% (w/w) in the dried hydrogel, such as in the range of 5-10% (w/w).Also combinations of these cryoprotectants and ranges may be used.

In one embodiment the content of the glycerol is in the range of 1-10%(w/w) and/or the content of the trehalose is in the range of 0.5-8%(w/w) in the dried hydrogel. In one embodiment the content of theglycerol is in the range of 5-10% (w/w) and/or the content of thetrehalose is in the range of 3-4% (w/w) in the dried hydrogel.

In one embodiment the content of the polyethylene glycol is in the rangeof 1-10% (w/w) and/or the content of the trehalose is in the range of0.5-8% (w/w) in the dried hydrogel. In one embodiment the content of thepolyethylene glycol is in the range of 5-10% (w/w) and/or the content ofthe trehalose is in the range of 3-4% (w/w) in the dried hydrogel.

The dried hydrogel may be provided as sheets, blocks or other shapes orforms, in general suitable for the desired purpose, and which may bethen rewetted or rehydrated before. The dried hydrogel may also beprovided as powder or in other crushed form. In such case the method ofpreparing the product may contain a step of forming the powder, forexample by grinding or crushing the freeze-dried product.

The obtained hydrogels, before or after drying, or more particularlyafter regelling, may be used in variety of applications, such as thosedescribed herein, for example in a method for providing, storing and/orculturing cells. The hydrogel may be provided for example as a medicalor a scientific product.

The rehydration or regelling may be carried out by providing aqueousrehydration liquid, which may contain further agents, preferablysterilized liquid. In one example the rehydration liquid comprises or iscell culture medium. The rehydration liquid may be provided as warmed,such as warmed to a temperature in the range of 30-40° C., such as about37° C. The dried hydrogel containing the cells may be contacted with theliquid and incubated for a period of time, such as for 10-60 minutes,before any further actions.

EXPERIMENTAL PART Cell Viability Assays

There is no simple in vitro test that could predict cell function afterfreezing in vivo. The cell viability assays must be specifically aimedat the components of each system that are damaged in the particularcircumstances under. Table 1 displays the main components of cellviability tracking. From these, six factors were chosen for theexperimental part of this work.

TABLE 1 Classification of Viability Assays. Highlighted methods werechosen as means of analysis for the experimental part of this work. I.Physical integrity 1. Gross (i) Appearance (ii) Physical property 2.Microscopic (i) Light microscopy (ii) Electron microscopy  (iii) CryoEMII. Metabolic activity 1. Uptake of metabolites 2. Production ofcatabolites 3. Labile metabolites 4. Enzymatic reactions (i)Intracellular  (ii) Membrane transport III. Mechanical activity 1.Motility 2. Phagocytosis 3. Contraction 4. Attachment 5. Aggregation IV.Mitotic activity 1. Mitotic index 2. DNA synthesis 3. “Plating” tests 4.Growth and development (i) Tissue culture  (ii) Embryonic V. Complete invivo function 1. Fertilization and development 2. Transplantation (i)Cells   (ii) Tissues  (iii) Vascularized organs

The gross appearance of the cells is typically observed with a lightmicroscopy unit at each step of the experiment. CryoEM can also be usedto image moist cell cultivations, however the sample is lost in theprocess, as with SEM imagining.

The metabolic activity of the cells can be quantified with theAlamarBlue® Cell Viability Assay, which is a non-toxic, stable andwater-soluble dye. The active ingredient of alamarBlue® reagent isresazurin which is blue in color, and nearly non-fluorescent. It is acell permeable compound, and inside cells reduced to resorufin, acompound that is red in color and highly fluorescent. This reductionincreases the overall red color and fluorescence of the mediasurrounding the cells. Hence, the cells can be monitored continuouslyand the increase in fluorescence measured as a function of time. Due tothis, the alamarBlue® assay has been considered superior to severalother assays for cell viability that required killing the cells, such asthe activity of cells oxidoreductase enzymes measuring MTT assay(3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, yellowtetrazol).

The active compound of alamarBlue® is resazurin, which is indigo blue incolor, and will enter the cytosol of cells when added to cell culturesamples by active intake. The mitochondrial enzyme activity converts itto resorufin by accepting electrons from NADH (the reduced form ofnicotinamide adenine dinucleotide) as visualized in FIG. 8. In additionto NADH, resorufin is also converted by accepting electrons fromnicotinamide adenine dinucleotide phosphate (NADPH), hydroquinone formof flavin adenine dinucleotide (FADH), reduced form of flavinmononucleotide (FMNH) and the cytochromes. The redox reaction changesthe color of the culture medium to fluorescent pink and this change canbe visually observed, but more importantly, accurately measured byfluorometric reading.

The alamarBlue® assay can also be implemented to measure the viabilityof 3D cell spheroid cultures directly in GrowDex®. In their work, theyused ultra-low attachment 96-well plates manufactured by Corning Inc. tocultivate the hydrogel-cell systems, and then used the alamarBlue® CellViability Reagent by Invitrogen as 1/10 of the co-volume of medium andhydrogel. As control samples, they used hydrogel systems without cells.

Another, yet more simple method to evaluate the cell viability is cellattachment. Many cell lines attach themselves to suitable surfaces invitro such as polypropylene or glass, when correct circumstances areapplied. The cells achieve this by excreting ECM proteins, such asfibronectin, collagen and laminin that are capable of sticking to roughsurfaces. Obviously, dead cells do not excrete these proteins and hencedo not actively attach themselves to these surfaces. Single cellstypically undergo apoptosis instead of attaching themselves, but in thecase of cancerous cell lines, this function has been disabled. Hence itcan be derived that cell attachment can be used as a viability assay oncancer cell lines on compatible surfaces.

One method to evaluate the cell membrane integrity is enzyme activityassays combined with nuclei staining. Calcein AM (acetoxy methyl) esterand propidium iodide are suitable for viability assessment of differentmicrobes. Calcein AM is itself a non-fluorescent and cell membranepermeable molecule, but undergoes an esterase reaction when in cytosolif correct enzymes are available and transforms into calcein, which is afluorescent molecule. This way, the fluorescent cells can be visuallyobserved with fluorescence microscopy or a plate reader.

Another aspect of the dual stain is propidium iodide (PI), a compoundthat is not cell membrane permeable and hence only enters the cytosol ifthe cell membrane has been damaged. Therefore, only necrotic cells areaffected, whereas apoptotic and healthy cells not. Once in the cytosol,the PI binds to any available DNA and RNA, including the DNA in nucleus.When excited with 535 nm light, an emission of 617 nm can be observed onthe areas of dead cells.

Rapid cooling was chosen as the freezing the method for the NFC-cellhydrogel systems. A droplet size of 20 μl was chosen for the step, wherethe NFC-cell hydrogel is in direct contact with liquid nitrogen. Forlyoprotection, trehalose and glycerol were used.

Diluted media was chosen as the rehydration fluid. Four factors werechosen for the determination of the cell viability. Cell attachment waschosen due to the overall picture on viability it provides, while cellmorphology is examined in each step with light microscopy. Resorufinoxidation was chosen as the method to evaluate the mitochondrialactivity of the spheroids in NFC, the fluorescent dual Cellstain todetermine the viability of the cells trough enzyme activity and cellmembrane integrity. Hep G2 cells were cultivated in GrowDex®.

The GrowDex® hydrogel forms a supporting matrix around the cells andprovides adjustable pressure and support that induces growth of 3D cellsystems. GrowDex® can be adjusted in several ways to fulfil therequirements of different cell types and to current date, GrowDex® hasbeen successfully optimized as a 3D cell culture matrix for sixdifferent types of cells; HepaRG, ARPE-19, MUG-Mel2 and Hep G2 celllines, and WA07 and iPS(IMR90)-4 stem cell lines.

Materials

1.5% (lot 11792) NFC hydrogel (GrowDex®) was obtained from UPM-KymmeneOyj, Finland. The diameter of most of the fibrils was in the range of4-10 nm and the length in the range of 500-10 000 nm. All othercompounds used were of analytical grade and either sterilized by UVlight or filtration prior to implementation. D-(+)-trehalose dehydrate,low adhesion 96-well inertGrade BRANDplates®, Cellstain double stainingkit, fetal bovine serum and glycerol (99%) were purchased fromSigma-Aldrich, USA. Polyethylene glycol (Mn 6000) was purchased fromFluka, Switzerland. Penicillin-streptomycin solution (PenStrep, 10 000U/ml), Dulbecco's modified eagle media (DMEM) without magnesium andcalcium and Dulbecco's Phosphate Buffered Saline (DPBS) 10× concentratewithout magnesium and calcium were purchased from Gibco, UK. Liquidnitrogen used in vitrification and argon gas used in preservation werepurchased from AGA Industrial Gases, Finland. MycoAlert™ MycoplasmaDetection Kit was bought from Lonza, Switzerland. PS SensoPlate™ 96 wellglass bottom plates for confocal imagining were purchased from GreinerBio-One, Austria. Sterile Corning® breathable sealing tape was purchasedfrom Corning, USA. The human hepatocellular carcinoma Hep G2 cells(passage number 100, ATCC HB-8065) were purchased from ATCC®, USA.TrypLE Express and alamarBlue® Cell Viability Reagent by Invitrogen waspurchased from Thermo Fisher Scientific. All solutions used were made indouble distilled ultrapure water.

Pulp obtained from birch tree is used as a material when producing theGrowDex® hydrogel. First, the pulp is purified and bleached. Next, thecellulose fibers are dispersed in double distilled ultrapure water andisolated in order to form a homogenous hydrogel. Lastly, the obtainedhydrogel is sterilized by autoclaving it in 121° C. for 20 minutes.

Methods

Cultivating 3D Cell Spheroids from Hep G2 Cancerous Liver Cell Line

Hep G2 cancerous cell line was selected for the experiments as it is awell characterized and a strong cell line, and has provided repeatableand reliable results in previous studies concerning cultivation in NFChydrogel. During the study, the cell line was cultivated with andwithout GrowDex® in order to obtain a control sample to differentiatethe effects of NFC in the survivability. 3D cell spheroids grown in 0.8%(w/w) GrowDex® are displayed in FIG. 1. Images (A), (B) and (C) displaytypical spheroids with a diameter between 100 and 120 μm. However, theimage (D) shows a larger spheroid with a diameter over 160 μm. Typicallythe spheroids do not grow to this large size in 5 days, implying thatthere was an cell aggregate at the seeding phase. The growth was limitedto 5 days, to avoid too large spheroids, as nutrients and oxygen cannotdiffuse to the core of a spheroid once it has reached a large sizeenough.

On top of 2D and 3D cultivations, an upkeep line was cultivated in cellflasks as a source of fresh cells for each new 2D and 3D seeding. Theupkeep bottle was split twice a week from passage numbers 100 to 152.The properties of Hep G2 cells have been studied to be unchangeable athigh passage numbers by ATCC. When the upkeep bottle was split for thepassage, the cells were first washed twice with 1×DPBS, detached with 4ml of tryple and reseeded to 75 cm² flasks with 11-14 ml of fresh media.The media contained 90% (v/v) of DMEM without calcium and magnesium and10% (v/v) of FBS for growth factors. In the 3D seeding phase, the cellswere spun in a centrifuge at 200 RCF (relative centrifugal force) for 6minutes and the formed supernatant was removed. Fresh media was added,and the cells were mixed with GrowDex® to create a 3D cell systemcontaining 0.8% (w/w) of NFC. An equal volume of fresh media was addedas a separate layer to provide nutrition and moisture. The cell plateswere then stored in an incubator at +37° C. and with 5% of CO₂ for 4days.

The cell medium (DMEM) contains phenol red, which gives a red color tothe media. Samples (B) and (C) in FIG. 1 were imaged without the medialayers (as they were vitrified without the media) resulting in a moreyellow background.

Optimization of Lyoprotectants

Three lyoprotectants were chosen for testing with GrowDex®. First, thecells were cultivated regularly for 4 days either in 2D or 3D with 0.8%(w/w) of GrowDex®, and then the medium was changed. For some of thesamples the medium was replaced with medium containing 50 mM oftrehalose dihydrate and possibly glycerol or PEG 6000, while othersreceived only regular medium. On day 5, the medium layer was mixed bypipetting with the hydrogel layer in order to form 0.4% (w/w) hydrogel.In the case of 2D cells, the cells were detached with triple prior tofreezing. All systems were frozen rapidly by injecting the mixture as 20μl droplets to liquid nitrogen. Then, the systems were freeze-dried andrehydrated and evaluated for viability with Cellstain double stainingand fluorescence microscopy. Table 2 demonstrates all of theoptimization combinations and their evaluated viabilities.

TABLE 2 Optimization chart for lyoprotectants and viabilities afterrehydration based on Cellstain double staining. PEG 6000 TrehaloseGlycerol Viability 0.7% 0.3% 1% (LIVE/ Lyoprotectants (w/w) (w/w) (w/w)DEAD) GrowDex ® 0.8% (w/w) & 24 h trehal. incub. x x x ++ GrowDex ® 0.8%(w/w) & 24 h trehal. incub. x x + GrowDex ® 0.8% (w/w) & 24 h trehal.incub. x + GrowDex ® 0.8% (w/w) & 24 h trehal. incub. x ++ GrowDex ®0.8% (w/w) & 24 h trehal. incub. x x ++ GrowDex ® 0.8% (w/w) & 24 htrehal. incub. + GrowDex ® 0.4% (w/w) & 24 h trehal. incub. x x x +++GrowDex ® 0.4% (w/w) & 24 h trehal. incub. x x ++ GrowDex ® 0.4% (w/w) &24 h trehal. incub. x ++ GrowDex ® 0.4% (w/w) & 24 h trehal. incub. x ++GrowDex ® 0.4% (w/w) & 24 h trehal. incub. x x +++ GrowDex ® 0.4% (w/w)& 24 h trehal. incub. ++ GrowDex ® 0.8% (w/w) x x + GrowDex ® 0.8% (w/w)x + GrowDex ® 0.8% (w/w) + Without GrowDex ® x x Without GrowDex ® x xWithout GrowDex ® x Without GrowDex ® & 24 h treha. Incub. x x WithoutGrowDex ® & 24 h treha. Incub. x x Without GrowDex ® & 24 h treha.Incub. x

Great results were obtained with the Cellstain from two combinations.The first mixture contained 0.4% (w/w) of GrowDex®, 1% (w/w) ofglycerol, 0.3% (w/w) of trehalose and it had been exposed to 50 mMtrehalose medium for 24 hours. The next mixture contained the samesubstances but in addition PEG 6000 (0.7% (w/w)). Therefore, it wasconcluded that PEG 6000 had most likely no or little role in thelyoprotection of the mixture. All the experiments from here on wereperformed with the first mixture (0.4% (w/w) of GrowDex®, 1% (w/w) ofglycerol, 0.3% (w/w) of trehalose and 24 h trehalose incubation).

Addition of the Lyoprotectants and Trehalose Incubation

After cultivating the cells for 4 days in 0.8% (w/w) GrowDex®, the abovelayer of media was changed. First, the cell systems were centrifuged at200 RCF for 6 minutes directly in the 96-well cultivation plates. Next,the supernatant was aspired from each well. Then, a new media mixturecontaining 50 mM of trehalose dihydrate, 2% (w/w) of glycerol and 1%(v/v) of PenStrep antibiotic mixture was added. The amount of glycerolwas double due to the fact that it was scaled against the medium layer,which is only half of the well. Next, the cells were incubated for 24hours at +37° C. and 5% CO₂. During the incubation, the cells in-tookpart of the trehalose, whilst the glycerol remained in the extracellularspace. After the incubation, the diameter of the grown spheroids wasmeasured with a Leica AF light microscope as shown in 10. Finally, themedia layer was mechanically mixed with the hydrogel layer by pipettingeach sample well back and forth. This ensured that the NFC content waslowered from 0.8% to approximately 0.4% thoroughly, which had earlierbeen optimized by us as the most suitable NFC amount for thelyoprotective matrix effect (lower concentration of fibers decreases theoverall porosity of the sample).

Differential Scanning Calorimetry

Freezing points (FP) and glass transition points (Tg) were measured fromthe hydrogel-cell mixtures with differential scanning calorimetry (DSC).The freeze-drying cycle in FIG. 5 was simulated with the DSC withcooling rate of 1° C./min and heating rate 1° C./10 min. In addition, acooling rate of 5° C./min was used to more distinctly point out the Tgtemperatures. Cell-hydrogels with cryoprotectants resulted in FP of−24.75° C. and in −23.75° C. without cells (control). The systemswithout cryoprotectants resulted in −22.77° C. with cells and in −22.35°C. without cells. Tg′ for the trehalose could not be detected even withthe accuracy of 0.2 mW, but this is most likely due to the low totalamount of trehalose in the samples.

Sterilization of Liquid Nitrogen

The liquid nitrogen provided by AGA, Finland, contains contaminants infrozen state, and as the cell samples are pipetted directly into theliquid nitrogen for maximum freezing rate in the vitrification step, theliquid nitrogen had to be sterilized before use. First, the liquidnitrogen was poured into a clean bucket as in FIG. 2 (A) and placedinside an ESCO laminar (DDTC/AU 550305054). Here, (B) it was sterilizedby ultraviolet (UV) light for 18 minutes with a dose of 20,000 μW/cm² ofUV-C radiation. Next, a custom built metallic rack was submerged intothe sterile nitrogen and then 15 ml sterile open tubes (C) wereaseptically attached to the rack and hence submerged into the sterileliquid nitrogen. Then, a metal spoon (D) was used to fill the tubes withapproximately 5 ml of sterile liquid nitrogen. As the tubes containingliquid nitrogen were submerged in the bucket of nitrogen, evaporationwas unnoticeable during the whole working and transportation steps,giving us an ideal working environment for the vitrification step, whichwas also performed inside the cell laminar. The excessive amount ofliquid nitrogen around the tubes also facilitated the transportation ofthe cell tubes to the freeze-drier, as it kept its temperature well.

Vitrification

In the next step, the cell plates were brought inside the laminar. Each96-well containing 3D cell spheroid hydrogel were properly mixed bypipetting with low adhesion pipette tips. By mixing the media layer ineach well with the hydrogel layer, a new hydrogel mixture with 0.4%(w/w) fiber content was formed. Next, the new hydrogel was carefullypipetted as tiny droplets directly into liquid nitrogen inside the 15 mltubes as in FIG. 3 (A). This caused the droplets to freeze as smallspheres with approximately 2-5 ml diameter. The freezing rate could bevisually observed to some extent, as the used media contains phenol red,a compound that switches color from pink to yellow as soon as no liquidwater is present. The observed color change from pink to yellow wasinstant, indicating rapid freezing. Lastly, the rack with the cell tubeswas placed inside a freeze-drying chamber (B) while still containingsome liquid nitrogen to protect the samples from excessive heat energy.

Freeze-Drying

After vitrification, the freeze-drying process was immediately executed.It was performed in Biocenter 2, Helsinki with ScanVac CoolSafemanufactured by Labogene. The model does not have a separate coolingsystem for samples and no programmable main unit, so the temperature pertime could not be tracked. However, due to phenol red in the cell media,we could exclude the presence of any free water in the samples at thebeginning of the freeze-drying cycle, as in the case of yellow sample inFIG. 4 (A). The condenser was kept at −106° C. during the whole process.

The samples were transported and placed inside the freeze-drier afterthe vitrification so that the 15 ml tubes still contained 2-3 ml ofliquid nitrogen as in FIG. 3 (B). The freeze-drying cycled wasimmediately initialized, so that the still forming nitrogen gas wascollected by the vacuum pump. This ensured the minimum startingtemperature for our samples, as the samples typically warm up during thevacuum initializing phase. The vacuum chamber reached a vacuum level of0.001 mbar in approximately 15 minutes. The freeze-drying cycle wascontinued for 72 hours, during which the samples warmed at an unknownrate, as the instrument bears no temperature sensors. A thermographiccamera was used through the chamber glass, however, it only measured thesurface temperature of the chamber instead of the samples. Nevertheless,the observed yellow hue of the phenol red ensured the dryness of thesamples at the end as in FIG. 4 (B). The vacuum was slowly released withroom air during 5 minutes.

Storage and Recreation

The dried samples were stored in closed plastic tubes secured withParafilm® and placed inside a desiccator with dried silica balls andfilled with argon gas and stored in +4° C. After one day of storage, thesamples were aseptically reconstructed with designed rehydration liquid,which was specially designed for the rewetting of the dried NFC systems.In order to avoid the shock from the high concentration differences, anovel rehydration liquid was designed to contain extra media in additionto ultrapure water, so that when added excessively, the original osmoticpressure of the system is achieved. The novel recreation liquidcontained 1% (v/v) of PenStrep mixture, 79% (v/v) of autoclavedultrapure water and 20% (v/v) of media. The rehydration liquid was addedto the dried samples so that the liquids volume corresponded 125% (v/v)of the dry samples original wet volume, except 2.5% (v/v) was reduced tocorrespond to the dry volume of the sample. Hence, by adding 122.5%(v/v) of the recreation liquid, the original osmotic pressure wasachieved while 20% (v/v) of fresh extra medium was added. Beforeaddition of the rehydration liquid, it was warmed to +37° C. to ensure agentle contact with the cells. The liquid was added to the samples byinserting a loaded 1000 μl low adhesion tip inside the 15-ml tube, andrapidly mixed by pipetting. The cells were then placed immediately in anincubator at +37° C. and 5% CO₂ for 15 minutes prior any otherprocessing.

Novel Freeze-Drying Cycles Maintaining Sterility

During the research, two novel sterile freeze-drying methods wereproduced. The first freeze-drying cycle was optimized in TurkuUniversity of Applied Sciences for freeze-drying doses of 200 μl of cellhydrogel systems. The instrument was a versatile drier with programmableoperating system in Kupittaa, Turku. The operated model was EPSILON 1-6Dand manufactured by CHRIST. The instrument had automated andprogrammable oil-based cooling shelves and a controlled vacuum pump.Therefore, we were able to design a unique freeze-drying cycle for 3Dcell hydrogel samples in 96-wells. The final optimized cycle ispresented in FIG. 5.

However, the research was later moved to the second freeze-drier inViikki, Helsinki. This model was ScanVac CoolSafe by Labogene. The modeldid not have a separate cooling system and no programmable main unit,so, as a result no cycle could be optimized for it. However, it was usedto produce all the results of this study. The samples were vitrifiedwith liquid nitrogen and dried in 0.001 mbar pressure for 72 hours.

As both of the used instruments were located in contaminated room air,two new sterile drying methods were developed for this study. The firstsystem in FIG. 6 (A) shows a 235 cm² cell culturing flask (manufacturedby Corning®) cut in two pieces, which have been sterilized andreconnected later in a sterile cell laminar after insertion of a 96-wellplate, which has been marked with an arrow. The cap of the bottle hasbeen replaced with a fresh, sterile packed cap inside the laminar.

Next, the junction between the reconnected bottle pieces was madeairtight with 70% ethanol wiped Parafilm® and after removal from thelaminar, with regular air conditioning tape. The whole flask was thencarefully placed inside the freeze-drying chamber, frozen 1° C./minutein a controlled manner and freeze-dried. The 96-well plate contained thecell hydrogel systems, and could only exchange gas molecules through theextensive filter of the cap. Afterwards, the dried samples were testedfor contaminations and mycoplasma with MycoAlert™ Mycoplasma DetectionKit. No contaminations or mycoplasma were detected.

In FIG. 6 (B), a polypropylene tube is shown tightly closed with aSterile Corning® breathable sealing tape. As no freeze-drying proofsealing tape was on the market, the manufacturer of this tape (Corning,USA) was contacted and a free sample received. The test results revealedthat liquid nitrogen had no effect on the adhesive properties of thetape, and it filtered the air as no contaminants nor mycoplasma weredetected afterwards.

Mycoplasma and Sterility Tests

After vitrification, freeze-drying, storage and recreation, the sampleswere tested for mycoplasma. The recreated cell hydrogel systems werecollected and seeded into 25 cm² culture flasks with 5 ml of media andthen cultivated for 8 days. During this time, the cell bottles wereobserved daily for any changes in media color, scent and appearance ofany microscopic activity was observed. No microbiological activity orchanges in the media pH or scent were discovered. Next, the mediasamples were tested for mycoplasma with MycoAlert™ Mycoplasma DetectionKit. It also yielded negative results, and therefore it was concludedthat both the Corning® breathable sealing tape and the cell flask corkfilters managed to maintain the sterility of the samples duringfreeze-drying.

Live/Dead Double Staining for Cell Viability

The recreated cell systems were tested for viability with a Live/Deaddouble staining kit for mammalian cells. The amount of calcein AM andpropidium iodide were first optimized with healthy 3D Hep G2 cellcultivations and with 70% ethanol killed cell control samples. Next, thefreeze-dried and recreated samples were diluted with DPBS (withoutcalcium and magnesium) buffer so that the final NFC concentration was0.1%, as during the optimization it was observed that this concentrationof NFC or below interrupted the least with the dyes, but the presence ofsome NFC was required to stabilize the cells for imagining. Spheroidswere suspended in staining solution containing Calcein-AM 1:500(Cellstain double staining kit; Sigma Aldrich) and 50 μg/ml DAPI(Invitrogen, Carlsbad, Calif.) for 15 min at 37° C., and transferred ona Greiner Sensoplate™ glass bottom 96 well plate (Sigma Aldrich). Thefluorescent images were taken with Leica SP5 II HCA microscope using HCPL APO 10×/0.4 (air) and HC PL APO 20×/0.7 CS (air) objectives. Imaris8.4.1 software was used for acquisition of the images. The possibilityfor autofluorescence was excluded by dead control samples. First, thefreeze-dried and recreated control systems were killed with 70% ethanolin a 30-minute incubation, and then dyed with the same dyes as thesamples. No green fluorescence was observed in the control samples.

AlamarBlue® Cell Viability Assay

The mitochondrial metabolic activity of the Hep G2 cell spheroids wasdetermined before and after freeze-drying with an oxidation-reductionindicator, resazurin (alamarBlue® Cell Viability Reagent, manufacturedby Invitrogen). First, alamarBlue® was applied as 1/10 of the co-volumeof the medium and hydrogel mixture. Then, the cells were exposed to theresazurin for 4 hours at 37° C. in 5% CO₂. Next, the culture plates werecentrifuged for 7 minutes at 200 rcf, and 50 ml of the formedsupernatant (medium) was transferred from each culture well to a black96-wellplate. Lastly, the metabolite of the resazurin (fluorescentresorufin) was recorded with a plate reader (Varioskan Flash, ThermoFisher) using excitation at 560 nm and emission at 590 nm.

The measured absorbance was then used to determine the number of viablecells by comparing the measured values to similar samples with knowncell amount. A linear standard line was drawn from this data(y=0.004x+32.364, R²=0.98211), shown in FIG. 7.

Results Morphology of the Cells Remained Unchanged During Freeze-Drying

Recreated cell systems were imaged with Leica AF light microscope with×5 objective (FIG. 8 A), ×10 objective (B) and ×20 objective (C and D).Also, rehydrated systems that were reseeded to cell flasks were imagedwith the same microscope. The recreated systems resembled remarkably theoriginal cell cultivations, with no observations of ruptured spheroids.Moreover, the cell morphology appeared to be normal to Hep G2 3D cellcultures. In FIG. 8 (A), extra-large cell spheroids with approximately300 μm diameter can be observed and in (B), typically sized and shapedspheroids and in (C) red-shaped spheroids. All the different sizedspheroids have survived without noticeable rupturing nor collapsing. In(D) the morphology of individual cells can be observed. Alsomicrovesicles on the freeze-dried 3D cell spheroids of HepG2 cells weredetected.

SEM Images of the Aerogel

After freeze-drying, the dried samples were imaged with a FEI Quanta™250 Field Emission Gun Scanning Electron Microscope (FEG SEM) (FIGS. 13A-F). The aerogel structure of GrowDex® resembled a typical highlyporous cellulose aerogel. Cell spheroids of different sizes could beobserved encapsulated in the aerogel (marked with arrows in FIGS. 13(A), (C) and (E).

Attachment of the Cells

After rehydration, some of the 3D cell spheroids were reseeded to 75 cm²cell cultivation flasks with fresh medium. After 4 hours, attachment ofthe cell spheroids on the polypropylene surface of the flask wasobserved, as presented in FIG. 9. The attachment was strong enough toanchor the spheroid so that it survived the change of medium at the4-hour point, where the old medium was aspired and new media pipetted tothe flask. As the supportive ECM mimicking pressure from the NFC fibernetwork was gone, morphological changes back to 2D-like structures wasobserved. However, most spheroids did not attach to the surface duringthe 4-hour incubation, suggesting lost cell viability.

Cellstain Double Staining Kit for Cell Viability

Cellstain double staining kit (also known as Live/Dead dual staining) bySigma-Aldrich was used for the determination of cell viability. Theamount of active compounds required, calcein AM and propidium iodide,was first optimized in a trial set before the actual measurements. Itwas determined that 0.2% (v/v) of the provided calcein AM solute and0.1% (v/v) of propidium iodide solute diluted in 1×DPBS resulted inclear images with 50 000 cells per well with a 15-minute incubation at37° C. and 5% of CO₂.

The rehydrated and dual stained cell samples were imaged with ConfocalMicroscope Leica SP5 II HCA with the following settings: pinhole 420 μm,gain 480 and argon laser 25%. The used lasers were HeNe 633 nm/12 mW andDPSS 561 nm/20 mW and the used objects were HC PL APO 10×/0.4 (air) andHC PL APO 20×/0.7 CS (air). FIG. 10 shows a cell spheroid 1 hour afterrehydration, where (A) shows the channel from the PDSS 561 laser and (B)the channel from HeNe 633 laser. FIG. 10 (C) shows the overlay of thesetwo channels with a higher capture resolution.

Autofluorescence was ruled out by killed control samples that werestained and imaged at the same time showing no green fluorescence inidentical settings. The killed control samples were taken from the samerehydrated cell batch, but treated with 70% ethanol for 30 minutesbefore staining and imagining. Stack images were captured with theconfocal microscopy, and 3D models were created in order to analyze theviability rate from the survived cell spheroids. Based on these models,a rate of 25% (±10) cell viability was derived as a result.

Another type of control samples was created to differ the lyoprotectiveeffect of GrowDex®; samples without it, that were otherwise treated thesame way including the 24 hour trehalose incubation, same amount oflyoprotectants and freezing with liquid nitrogen. These control sampleswere freeze-dried with the main samples at the same time to rule outdifferences between batches. The results are presented in FIG. 11, whereall the control samples in (A) were found dead, but live cells wereobserved in the main samples in (B).

The largest cell spheroids appeared to be hollow in some cases. Thismight be due to the fact, that the stains were only given 15 minutestime to stain the spheroids and the stains need to diffuse trough theouter cell layers towards the core. This might create an illusion ofhollow spheroids, where in reality the stain incubation time should beincreased, or the spheroids cut in half before staining. A middle partof a hollow-appearing spheroid is presented in FIG. 12.

With the live/dead images, exact classification to dead an alive ischallenging. Some individual cells had both stains, meaning holes intheir cell membrane, but enzyme activity inside the cells. Nevertheless,the experiment clearly proves the lyoprotective effects of GrowDex®.

AlamarBlue® Assay

After freeze-drying, the viability of the Hep G2 cells was measured withalamarBlue® assay resulting in 3.7% (±0.03) viability, which isrelevantly low. Nevertheless, the viability was detected in severalmeasurement sets, and the alamarBlue® kit is sensitive enough to detecteven 50 viable cells per well. This is still controversy to the resultswith the Cellstain double staining. One theory was that thefreeze-drying changes the properties of GrowDex® and makes it sticky toresazurin. However, this was ruled out by control sample sets, whereGrowDex® was first freeze-dried with medium and lyoprotectants butwithout cells, and healthy cells were seeded after rehydration.Nevertheless, these cells showed normal viability

The lack of viability in the alamarBlue® activity measurements provesthat something happens to the cell mitochondria during thefreeze-drying. Nevertheless, the cell membrane stays intact and cellenzyme proteins stay active as proven by several repeated measurementssets of Cellstain double staining. During the problematic discussion, wereferred to the cells as Schrödinger's cells. As both assays wereimplemented numerous times with always similar results, and all otherscenarios were ruled out by comprehensive control measurements, wefinally came to the conclusion that the cell membrane remains relevantlyintact as no green dye is leaking, while the mitochondria inside thecells are damaged to some extent. One could argue that the NFC preventsthe green stain from leaking, but this cannot be the case, as itdiffuses from the NFC to the cells in the first place. This dilemma ofalamarBlue® is discussed in more detail in the next section.

The nanofibrillar cellulose hydrogels are useful as lyoprotectivematrices that can withstand freezing and drying without mechanicalrupture, are inert, are clean of contaminants, hold enough liquid toachieve high porosity and can be shaped as well-defined geometricalunits. According to the tests, the nanofibrillar cellulose produced byUPM-Kymmene Oyj fulfils all of these requirements. The NFC matrix didrelease the absorbed active product when flooded with the rehydrationliquid. If a correct amount of cellulase enzyme mixture (GrowDase™) isadded, the nanofibrillar cellulose fiber structure is digested to smallsugars. However, in the case of freeze-dried 3D cell spheroid hydrogelsystems, the cultivation is most likely continued in the hydrogelenvironment, and therefore there is usually no need to digest thehydrogel in the first place.

As to the requirements for freeze-dried products that the dried productshould be dry, active, shelf stable, clean and sterile, ethicallyacceptable, pharmaceutically elegant, readily soluble and simple toreconstitute and the process should be economically practicable, alsothese requirements were met. Based on the findings, the dried GrowDex®Hep G2 system had no free water left in it, retained its activity afterreconstitution, was sterile, elegant and readily soluble. As theGrowDex® and all other components are completely animal free, theprocess should be considered ethically acceptable, as long as the usedcells have no ethical issues. If the required machinery is available,all materials used in the process are relevantly inexpensive (sugars,cellulose, glycerol), yet the final product can be of high value. If theprocess is further optimized in factory settings, it can be consideredas economically practicable. The shelf stableness of the product was notresearched beyond days due to tight schedule. Also, the freeze-driedsamples were transferred in dry state encapsulated in NFC fromWest-Finland to Helsinki in a car trunk on a highway—yet the samplesstill showed viability. It could be due to the rigid dry structure ofthe NFC fibers that prevent the hollow cells from collapsing.

1. A method for freeze-drying extracellular vesicles in a hydrogelcomprising nanofibrillar cellulose, the method comprising: providing ahydrogel comprising nanofibrillar cellulose, providing extracellularvesicles, combining the extracellular vesicles and the hydrogelcomprising nanofibrillar cellulose to form a vesicle system, and freezedrying the vesicle system to obtain freeze-dried extracellular vesiclesin an aerogel comprising freeze-dried nanofibrillar cellulose.
 2. Themethod of claim 1, wherein the extracellular vesicles comprise exosomesor microvesicles.
 3. The method of claim 1, further comprising providingone or more cryoprotective agent(s) and/or one or more lyoprotectiveagent(s), and adding the one or more cryoprotective agent(s) and/or oneor more lyoprotective agent(s) to the vesicle system.
 4. The method ofclaim 3, wherein the one or more cryoprotective and/or lyoprotectiveagent(s) is selected from trehalose, glycerol, and polyethylene glycol.5. The method of claim 3, wherein the one or more cryoprotective and/orlyoprotective agent(s) comprise trehalose and glycerol.
 6. The method ofclaim 1, wherein the nanofibrillar cellulose has a number average fibrildiameter of 1 to 100 nanometers.
 7. The method of claim 1, wherein thenanofibrillar cellulose, when dispersed in water, provides a zero-shearviscosity of 5000-50000 Pas and a yield stress of 3-15 Pa, determined byrotational rheometer at a consistency of 0.5% (w/w) in aqueous medium at22° C.
 8. The method of claim 1, wherein the concentration of thenanofibrillar cellulose in the hydrogel before the freeze-drying is inthe range of 0.1-10% (w/w), such as 0.1-5% (w/w), 0.1-2% (w/w), or0.1-1.0% (w/w).
 9. The method of claim 1, wherein the nanofibrillarcellulose is selected from anionically modified nanofibrillar cellulose,cationically modified nanofibrillar cellulose and unmodifiednanofibrillar cellulose, and TEMPO oxidized nanofibrillar cellulose. 10.The method of claim 1, wherein the freeze drying is continued until thedried hydrogel has a moisture content of 10% (w/w) or less, preferablyin the range of 1-10% (w/w), such as 2-8% (w/w).
 11. A freeze-driedaerogel comprising freeze-dried nanofibrillar cellulose and freeze-driedextracellular vesicles, wherein the moisture content of the hydrogel is10% (w/w) or less, preferably in the range of 1-10% (w/w), such as 2-8%(w/w).
 12. The freeze-dried aerogel of claim 11, wherein theextracellular vesicles comprise exosomes or microvesicles.
 13. Thefreeze-dried aerogel of claim 11, further comprising one or morecryoprotective agent(s) and/or one or more lyoprotective agent(s). 14.The freeze-dried aerogel of claim 13, wherein the one or morecryoprotective and/or lyoprotective agent(s) is selected from trehalose,glycerol, and polyethylene glycol.
 15. The freeze-dried aerogel of claim13, wherein the one or more cryoprotective and/or lyoprotective agent(s)agents comprise trehalose and glycerol.
 16. The freeze-dried aerogel ofclaim 11, wherein the content of the extracellular vesicles in thefreeze-dried aerogel is in the range of 0.1-65% (w/w), such as in therange of 0.1-50% (w/w), for example in the range of 1-25% (w/w).
 17. Thefreeze-dried aerogel of claim 13, wherein the content of the one or morecryoprotective and/or lyoprotective agent(s) is in the range of 1-10%(w/w) and/or the content of the trehalose is in the range of 0.5-50%(w/w) in the freeze-dried aerogel.
 18. The freeze-dried aerogel of claim11, wherein the nanofibrillar cellulose has a number average fibrildiameter of 1 to 100 nanometers.
 19. The freeze-dried aerogel of claim11, wherein the nanofibrillar cellulose, when dispersed in water,provides a zero-shear viscosity of 5000-50000 Pa·s and a yield stress of3-15 Pa, determined by rotational rheometer at a consistency of 0.5%(w/w) in aqueous medium at 22° C.
 20. The freeze-dried aerogel of claim11, wherein the nanofibrillar cellulose is selected from anionicallymodified nanofibrillar cellulose, cationically modified nanofibrillarcellulose and unmodified nanofibrillar cellulose, and TEMPO oxidizednanofibrillar cellulose.
 21. The freeze-dried aerogel of claim 11,obtained with the method of the claim 1.